44
A. Meyerdierks and F.O. Glöckner
3 kb insert size (Zhang et al. 2006). Increased sequencing effort and iterative assembly strategies have also been applied to overcome this problem (Zhang et al. 2006).
The second issue, the amplification bias observed for MDA reactions (Dean et al.
2002, Hosono et al. 2003, Abulencia et al. 2006, Yokouchi et al. 2006) is of importance when the input is reduced to several or only one microbial cell (Raghunathan
et al. 2005, Zhang et al. 2006, Kvist et al. 2007, Podar et al. 2007). As this bias
seems to be sequence independent, this limitation of the method may be overcome
by deep sequencing, and the combination of multiple MDA reactions from the same
DNA sample (Raghunathan et al. 2005, Abulencia et al. 2006, Zhang et al. 2006).
Non-specific DNA amplification is especially observed when minute amounts of
DNA are amplified, and might originate from primer dimers or trace contaminants
of DNA (Raghunathan et al. 2005, Zhang et al. 2006). MDA has been used in several
studies for whole genome amplification (WGA) of single uncultured microorganisms, isolated from environmental samples by single cell isolation methods
(Zhang et al. 2006, Kvist et al. 2007, Marcy et al. 2007, Stepanauskas and Sieracki
2007).
When a mixed sample of eight bacterial genomes of different sizes and G+C
contents was amplified by MDA a significant bias towards the amplification of certain strains was observed (Abulencia et al. 2006). A case study with low biomass
samples from contaminated soils revealed only small changes in the taxonomic
groups detected, and a slightly higher number of species were inferred based on 16S
rRNA libraries constructed from MDA treated community DNA (Abulencia et al.
2006). An investigation of microbial communities in the deep biosphere revealed
a similar phylogenetic and functional category distribution in MDA amplified and
non-amplified samples. However, the phylogenetic distribution of 16S rRNA genes
and ribosomal proteins differed significantly in this study, probably due to the low
number of such genes in the dataset (Biddle et al. 2008). An evaluation of the MDAassociated bias using 16S rRNA denaturing gradient gel electrophoresis (DGGE)
fingerprints revealed a slight amplification bias when the DNA input in the MDA
reaction was below one nanogram (Neufeld et al. 2008). MDA-amplified DNA has
already been successfully used to construct a large insert fosmid library from heavy
DNA retrieved from a stable isotope probing experiment. Chimeric artefacts were
observed at the ends and within a fosmid insert, making a verification of the gene
order by PCR necessary. Nevertheless, the approach represents a significant step
forward in the assessment of the genetic potential, including complete operon structures, of uncultured, active microorganisms in marine surface waters (Neufeld et al.
2008).
Other studies in which the amplification of metagenomic DNA was a prerequisite
for the study of uncultured marine microbes include a study of marine viral assemblages (Angly et al. 2006), an analysis of the genetic potential of two multicellular
filaments of uncultured, marine, large sulfur bacteria (Mussmann et al. 2007), and an
analysis of marine methanotrophic microbial consortia, isolated by a combination
of fluorescence in situ hybridisation and magnetic bead capture (Pernthaler et al.
2008).
A. Meyerdierks and F.O. Glöckner
3 kb insert size (Zhang et al. 2006). Increased sequencing effort and iterative assembly strategies have also been applied to overcome this problem (Zhang et al. 2006).
The second issue, the amplification bias observed for MDA reactions (Dean et al.
2002, Hosono et al. 2003, Abulencia et al. 2006, Yokouchi et al. 2006) is of importance when the input is reduced to several or only one microbial cell (Raghunathan
et al. 2005, Zhang et al. 2006, Kvist et al. 2007, Podar et al. 2007). As this bias
seems to be sequence independent, this limitation of the method may be overcome
by deep sequencing, and the combination of multiple MDA reactions from the same
DNA sample (Raghunathan et al. 2005, Abulencia et al. 2006, Zhang et al. 2006).
Non-specific DNA amplification is especially observed when minute amounts of
DNA are amplified, and might originate from primer dimers or trace contaminants
of DNA (Raghunathan et al. 2005, Zhang et al. 2006). MDA has been used in several
studies for whole genome amplification (WGA) of single uncultured microorganisms, isolated from environmental samples by single cell isolation methods
(Zhang et al. 2006, Kvist et al. 2007, Marcy et al. 2007, Stepanauskas and Sieracki
2007).
When a mixed sample of eight bacterial genomes of different sizes and G+C
contents was amplified by MDA a significant bias towards the amplification of certain strains was observed (Abulencia et al. 2006). A case study with low biomass
samples from contaminated soils revealed only small changes in the taxonomic
groups detected, and a slightly higher number of species were inferred based on 16S
rRNA libraries constructed from MDA treated community DNA (Abulencia et al.
2006). An investigation of microbial communities in the deep biosphere revealed
a similar phylogenetic and functional category distribution in MDA amplified and
non-amplified samples. However, the phylogenetic distribution of 16S rRNA genes
and ribosomal proteins differed significantly in this study, probably due to the low
number of such genes in the dataset (Biddle et al. 2008). An evaluation of the MDAassociated bias using 16S rRNA denaturing gradient gel electrophoresis (DGGE)
fingerprints revealed a slight amplification bias when the DNA input in the MDA
reaction was below one nanogram (Neufeld et al. 2008). MDA-amplified DNA has
already been successfully used to construct a large insert fosmid library from heavy
DNA retrieved from a stable isotope probing experiment. Chimeric artefacts were
observed at the ends and within a fosmid insert, making a verification of the gene
order by PCR necessary. Nevertheless, the approach represents a significant step
forward in the assessment of the genetic potential, including complete operon structures, of uncultured, active microorganisms in marine surface waters (Neufeld et al.
2008).
Other studies in which the amplification of metagenomic DNA was a prerequisite
for the study of uncultured marine microbes include a study of marine viral assemblages (Angly et al. 2006), an analysis of the genetic potential of two multicellular
filaments of uncultured, marine, large sulfur bacteria (Mussmann et al. 2007), and an
analysis of marine methanotrophic microbial consortia, isolated by a combination
of fluorescence in situ hybridisation and magnetic bead capture (Pernthaler et al.
2008).