Genomic DNA Amplification from a Single Bacterium

Raghunathan, Arumugham; Ferguson, Harley R.; Bornarth, Carole J.; Song, Wanmin; Driscoll, Mark D.; Lasken, Roger S.

doi:10.1128/aem.71.6.3342-3347.2005

Cited by 276 publications

(273 citation statements)

References 7 publications

Supporting

Mentioning

268

Contrasting

Unclassified

Order By: Relevance

“…The purpose of the lysis step is to degrade the microbial cell envelope without damaging the chromosomal DNA, introducing contamination or inhibiting subsequent amplification reactions. The majority of single-cell studies rely on an alkaline lysis procedure first described by Raghunathan et al 21 , although alternative or supplementary treatments such as heat, freeze-thaw, detergents and treatment with hydrolytic enzymes have been used 6 . However, these lysis techniques are not universally effective on all microbes, and thus a substantial fraction of genomes may not be recovered from some environmental samples.…”

Section: Experimental Designmentioning

confidence: 99%

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

et al. 2014

View full text Add to dashboard Cite

single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by Facs. the protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16s rrna-based identification of individual bacterial and archaeal cells. the described procedure can be performed with standard molecular biology equipment and a Facs machine. It takes <12 h of bench time over a 4-d time period, and it generates up to 1 mg of genomic Dna from an individual microbial cell, which is suitable for downstream applications such as pcr amplification and shotgun sequencing. the completeness of the recovered genomes varies, with an average of ~50%.

show abstract

Section: Experimental Designmentioning

confidence: 99%

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Hutchison et al (2005) discovered that a smaller MDA reaction volume (3 ml) greatly reduced the amount of nonspecific DNA generated with 50 molecules of singlestranded circular M13 template compared to higher reaction volumes (15 or 30 ml). Others have shown that the total number of DNA templates affects representational bias (Lasken et al, 2005), with a single copy of template giving the highest bias (Raghunathan et al, 2004). Reducing bacterial DNA template from 10 ng (410 6 genome copies) by 10-and 100-1000-fold reduced representational bias 3-and 6-fold, respectively (Wu et al, 2006).…”

Section: A Brief History Of Mdamentioning

confidence: 99%

“…Progress has been made in the ability to analyze genomes, without the prerequisite of cultivation, by using MDA from individual cells. The first demonstration of genomic sequencing from single cells used E. coli isolated by fluorescence-activated cell sorting, followed by MDA (Raghunathan et al, 2004). Amplification of B5 fg of template generated B24 mg of product, more than a billion-fold amplification.…”

Section: Use Of Mda In Microbial Ecologymentioning

confidence: 99%

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

2008

View full text Add to dashboard Cite

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.

show abstract

“…However, MDA from a single cell suffers from amplification bias. The single chromosome copy is broken into DNA fragments, further adding to amplification bias, and any contaminating DNA can also contribute to the sequences obtained (Raghunathan et al 2005;Woyke et al 2011). It is therefore generally not possible to finish a microbial genome starting from a single cell (Rodrigue et al 2009).…”

mentioning

confidence: 99%

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

Fitzsimons

Novotny

et al. 2013

Genome Res.

Self Cite

View full text Add to dashboard Cite

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

show abstract

Genomic DNA Amplification from a Single Bacterium

Cited by 276 publications

References 7 publications

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

Contact Info

Product

Resources

About