Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Woyke, Tanja; Sczyrba, Alexander; Lee, Janey; Rinke, Christian; Tighe, Damon; Clingenpeel, Scott; Malmstrom, Rex R.; Stepanauskas, Ramunas; Cheng, Jan‐Fang

doi:10.1371/journal.pone.0026161

Cited by 161 publications

(165 citation statements)

References 15 publications

Supporting

Mentioning

150

Contrasting

Order By: Relevance

“…This isothermal, strand-displacing amplification yields, on average, >10-kb-long overlapping amplicons, which are suitable for whole-genome sequencing and de novo assembly, similarly to sequence data from DNA extracts of pure cultures. However, MDA results in uneven genome coverage that can be partially mitigated by wet-bench and bioinformatic normalization methods 1,20,23,24 . Genomic rearrangements, or chimeras, are formed during MDA and can complicate genome assembly by linking noncontiguous chromosomal regions 15,25 .…”

Section: Experimental Designmentioning

confidence: 99%

“…MDA reactions are sensitive to DNA contamination present in the sample, reagents or contamination introduced through sample handling. The effect of DNA contamination can be controlled by establishing clean cell-sorting procedures 12,20,24,26 , decontaminating commercially available MDA reagents 1,20,23 , expressing a custom ultrapure Phi29 enzyme 27 or by using commercial precleaned MDA kits that are specifically designed for single-cell templates. Volume reduction can also limit the impact of reagent contamination (Box 1).…”

Section: Experimental Designmentioning

confidence: 99%

“…20| UV-treat the tubes of master mix in a reflective container on ice for 30-90 min in the Spectraline UV cross-linker, as described previously 23 . Arrange the tubes to rest ~8 cm below the UV bulb.…”

Section: | To Each Well (Of the Plates Frommentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2014

Self Cite

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%

Section: Experimental Designmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…The genomic DNA from both samples was subjected to whole-genome multiple displacement amplification (MDA) using the REPLI-g Mini kit (Qiagen) according to the manufacturer's recommendations. A UV irradiation method was employed to eliminate exogenous contaminant DNA found in MDA reagents (Woyke et al, 2011) and 25 independent amplification reactions were pooled for further analysis. Before genome sequencing, amplified DNA (B20 mg) was purified using the QIAamp DNA Mini kit (Qiagen) following the manufacturer's supplementary protocol.…”

Section: Sample Collectionmentioning

confidence: 99%

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

Goffredi

Zhang

et al. 2013

The ISME Journal

View full text Add to dashboard Cite

An unusual symbiosis, first observed at B3000 m depth in the Monterey Submarine Canyon, involves gutless marine polychaetes of the genus Osedax and intracellular endosymbionts belonging to the order Oceanospirillales. Ecologically, these worms and their microbial symbionts have a substantial role in the cycling of carbon from deep-sea whale fall carcasses. Microheterogeneity exists among the Osedax symbionts examined so far, and in the present study the genomes of the two dominant symbionts, Rs1 and Rs2, were sequenced. The genomes revealed heterotrophic versatility in carbon, phosphate and iron uptake, strategies for intracellular survival, evidence for an independent existence, and numerous potential virulence capabilities. The presence of specific permeases and peptidases (of glycine, proline and hydroxyproline), and numerous peptide transporters, suggests the use of degraded proteins, likely originating from collagenous bone matter, by the Osedax symbionts. 13 C tracer experiments confirmed the assimilation of glycine/ proline, as well as monosaccharides, by Osedax. The Rs1 and Rs2 symbionts are genomically distinct in carbon and sulfur metabolism, respiration, and cell wall composition, among others. Differences between Rs1 and Rs2 and phylogenetic analysis of chemotaxis-related genes within individuals of symbiont Rs1 revealed the influence of the relative age of the whale fall environment and support possible local niche adaptation of 'free-living' lifestages. Future genomic examinations of other horizontally-propogated intracellular symbionts will likely enhance our understanding of the contribution of intraspecific symbiont diversity to the ecological diversification of the intact association, as well as the maintenance of host diversity.

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.

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

Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Cited by 161 publications

References 15 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

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

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

Contact Info

Product

Resources

About