Binga EK, Lasken RS, Neufeld JD.. Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology. ISME J 2: 233-241

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
The ISME Journal (Impact Factor: 9.3). 04/2008; 2(3):233-41. DOI: 10.1038/ismej.2008.10
Source: PubMed


Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a 'power tool' in the molecular toolbox of microbial ecologists.

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    • "Care must be taken in interpreting diversity estimates calcu - lated from the data . First , evenness can be influenced by the use of MDA , which can unevenly amplify the initial template ( Dean et al . , 2001 ) , be biased toward small circular ssDNA molecules and form chimeras ( Binga et al . , 2008 ; Polson et al . , 2011 ) ."
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    ABSTRACT: Metagenomic studies have revealed that ssDNA phages from the family Microviridae subfamily Gokushovirinae are widespread in aquatic ecosystems. It is hypothesized that gokushoviruses occupy specialized niches, resulting in differences among genotypes traversing water column gradients. Here, we use degenerate primers that amplify a fragment of the gene encoding the major capsid protein to examine the diversity of gokushoviruses in Saanich Inlet (SI), a seasonally anoxic fjord on the coast of Vancouver Island, BC, Canada. Amplicon sequencing of samples from the mixed oxic surface (10 m) and deeper anoxic (200 m) layers indicated a diverse assemblage of gokushoviruses, with greater richness at 10 m than 200 m. A comparison of amplicon sequences with sequences selected on the basis of RFLP patterns from eight surface samples collected over a 1-year period revealed that gokushovirus diversity was higher in spring and summer during stratification and lower in fall and winter after deep-water renewal, consistent with seasonal variability within gokushovirus populations. Our results provide persuasive evidence that, while specific gokushovirus genotypes may have a narrow host range, hosts for gokushoviruses in SI consist of a wide range of bacterial taxa. Indeed, phylogenetic analysis of clustered amplicons revealed at least five new phylogenetic groups of previously unknown sequences, with the most abundant group associated with viruses infecting SUP05, a ubiquitous and abundant member of marine oxygen minimum zones. Relatives of SUP05 dominate the anoxic SI waters where they drive coupled carbon, nitrogen, and sulfur transformations along the redoxline; thus, gokushoviruses are likely important mortality agents of these bacteria with concomittant influences on biogeochemical cycling in marine oxygen minimum zones.
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    • "Gene-targeted sequencing based on PCR amplification technique is able to analyse the minute amounts of template DNA recovered in forensic samples. The need for a relatively large amount of initial DNA template for shotgun sequencing makes this approach less suitable for forensic oriented metagenomic analysis but whole-genome amplification (WGA), using Phi 29 DNA polymerase, represents an effective way of enabling whole-genome shotgun sequencing from small quantities of DNA [29]. "
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    ABSTRACT: Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "This breakthrough was enabled by the development of the MDA reaction (Dean et al., 2001, 2002), which can amplify a single genome copy more than a billion fold enabling sequencing of DNA from very low (femtogram) levels (about the amount of DNA in a single bacterial cell). Bacteria that have not been cultivated by conventional culturing techniques are currently the central target of single-cell genomics (Lasken et al., 2005; Raghunathan et al., 2005; Hutchison and Venter, 2006; Ishoey et al., 2008; Lasken, 2012) The recent advancements in DNA sequencing of single bacterial cells has accelerated the study of uncultivated microbes (Lasken, 2012), providing genomic assemblies for species previously known only from 16S rRNA clone libraries and metagenomic data (Marcy et al., 2007; Podar et al., 2007; Binga et al., 2008; Eloe et al., 2011; Youssef et al., 2011; Dupont et al., 2012; McLean et al., 2013a; Nurk et al., 2013b; Rinke et al., 2013). Using these approaches, the so-called “dark matter of life” which represents uncultivated microbes and even entire divisions of bacterial phyla (candidate divisions and candidate phyla) are slowly being revealed with assembled genomes. "
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    ABSTRACT: Oral microbes represent one of the most well studied microbial communities owing to the fact that they are a fundamental part of human development influencing health and disease, an easily accessible human microbiome, a highly structured and remarkably resilient biofilm as well as a model of bacteria-bacteria and bacteria-host interactions. In the last 80 years since oral plaque was first characterized for its functionally stable physiological properties such as the highly repeatable rapid pH decrease upon carbohydrate addition and subsequent recovery phase, the fundamental approaches to study the oral microbiome have cycled back and forth between community level investigations and characterizing individual model isolates. Since that time, many individual species have been well characterized and the development of the early plaque community, which involves many cell-cell binding interactions, has been carefully described. With high throughput sequencing enabling the enormous diversity of the oral cavity to be realized, a number of new challenges to progress were revealed. The large number of uncultivated oral species, the high interpersonal variability of taxonomic carriage and the possibility of multiple pathways to dysbiosis pose as major hurdles to obtain a systems level understanding from the community to the gene level. It is now possible however to start connecting the insights gained from single species with community wide approaches. This review will discuss some of the recent insights into the oral microbiome at a fundamental level, existing knowledge gaps, as well as challenges that have surfaced and the approaches to address them.
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