Whole Genome Amplification and De novo Assembly of Single Bacterial Cells

Rodrigue, Sébastien; Malmstrom, Rex R.; Berlin, Aaron M.; Birren, Bruce W.; Henn, Matthew R.; Chisholm, Sallie W.

doi:10.1371/journal.pone.0006864

Cited by 237 publications

(258 citation statements)

References 32 publications

(66 reference statements)

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“…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%

“…Genomic rearrangements, or chimeras, are formed during MDA and can complicate genome assembly by linking noncontiguous chromosomal regions 15,25 . The effect of these rearrangements can be limited by high sequence coverage and by avoiding long mate-pair libraries 24 .…”

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%

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

et al. 2014

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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%.

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Section: Experimental Designmentioning

confidence: 99%

Section: Experimental Designmentioning

confidence: 99%

Section: Experimental Designmentioning

confidence: 99%

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

et al. 2014

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“…Sample collection tubes and the sheath fluid reservoir are treated with UV for 2 hours before filling (Rodrigue, et al, 2009). Sheath fluid may be as simple as a 1% NaCl solution prepared with UV-treated ddH 2 0 and heat combusted NaCl (450°C for 4 hours), or a commercially available formula such as BioSure (BioSure, Grass Valley, CA USA).…”

Section: Preparation For Sortingmentioning

confidence: 99%

“…Sheath fluid may be as simple as a 1% NaCl solution prepared with UV-treated ddH 2 0 and heat combusted NaCl (450°C for 4 hours), or a commercially available formula such as BioSure (BioSure, Grass Valley, CA USA). Sheath fluid and sample lines are cleaned by running warm water followed by bleach solution (5-10%) and extensive flushing with UV-treated, DNA-free ddH 2 0 (Stepanauskas & Sieracki, 2007;Rodrigue, et al, 2009). …”

Section: Preparation For Sortingmentioning

confidence: 99%

Coupling FACS and Genomic Methods for the Characterization of Uncultivated Symbionts

Thompson

Bench

Carter

et al. 2013

Methods in Enzymology

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Symbioses between microbes are likely widespread and functionally relevant in diverse biological systems however they are difficult to discover. Most microbes remain uncultivated, symbioses can be relatively rare or dynamic, and intercellular connections can be delicate. Thus traditional methods such as microscopy are inadequate for efficient discovery and precise characterization of novel interactions, their metabolic basis, and the species involved. Highthroughput metagenomic sequencing of entire microbial communities has revolutionized the field of microbial ecology, however genomic signals from symbionts can get buried in sequences from abundant organisms and evidence for direct links between microbial species cannot be gained from bulk samples. Thus, a specialized approach to the characterization of symbioses between naturally-occurring microbes is required. This chapter presents methods for combining fluorescence-activated cell sorting (FACS) to isolate and separate uncultivated symbionts with molecular biology techniques for DNA amplification in order to characterize uncultivated symbionts through genomic and metagenomic techniques.

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