Single cell genome sequencing

Yilmaz, Suzan; Singh, Anup K.

doi:10.1016/j.copbio.2011.11.018

Cited by 90 publications

(59 citation statements)

References 45 publications

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“…Major chemistries employed in the singlecell WGA include the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), the multiple displacement amplification (MDA), the multiple annealing and looping-based amplification cycles (mALBAC) and the ligationmediated PCR following a site specific DNA digestion as in the case of this study (4,17,19,20,(25)(26)(27). However, besides the operator capability, all these strategies are labor-intensive, require long time of execution and may imply different degrees of errors due to inappropriate starting in the early stages of amplification, or due to the specific chemicals used in the reaction (17,18,28).…”

Section: Discussionmentioning

confidence: 99%

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

Palmirotta

Lovero

Silvestris

et al. 2017

CGP

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Section: Discussionmentioning

confidence: 99%

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

Palmirotta

Lovero

Silvestris

et al. 2017

CGP

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“…Although sorting by FCM was shown to efficiently purely separate and isolate the target cells at the single-cell level (see Table S5 in the supplemental material), there are several known difficulties in WGA of a single bacterial cell with respect to the efficiency of genome amplification and purity of the products (38)(39)(40). The amplification efficiency is known to be low, and the successful amplification efficiency of the 16S rRNA gene with WGA products from a single bacterial cell is 30% at most (41).…”

Section: Discussionmentioning

confidence: 99%

Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

Shintani

Matsui

Inoue

et al. 2014

Appl Environ Microbiol

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The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria, Bacteroidetes, and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a "transient" host of pCAR1. P lasmids are circular or linear extrachromosomal replicons, which are often transmissible by conjugation (1, 2). Plasmids can spread among bacteria effectively and act as key "vehicles" of pathogenicity and environmentally relevant traits (1). Therefore, the conjugative transfer of plasmids is one of the most important mechanisms to promote the rapid evolution and adaptation of bacteria. Knowledge about the host range of a plasmid is essential to the understanding of how the plasmid is transferred in different environments. A plasmid host is generally recognized in two ways: (i) cells can obtain the plasmid by conjugative transfer and (ii) cells can replicate and maintain the plasmid after cell division. Although some plasmids are known to make junctions to cross the kingdom barrier (3), to date, plasmid host ranges have been determined by using culture-dependent methods by detecting colonies of transconjugants on selective media. This method is unable to detect uncultivated or noncultivable hosts and is limited by both the transfer system and its replication and maintenance systems. Recently, several studies have shown that noncultivable bacteria carry plasmids (4, 5), suggesting that other uncultivated or noncultivable bacteria could also have plasmids. Because the majority of environmental bacteria are difficult to cultivate, determining an accurate host range for the plasmid transfer function (i.e., a transfer range of plasmids) has not yet been accomplishe...

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“…Also, as the current WGS approach relies on isolation of pure cultures, it is infeasible to be directly applied on clinical samples in which a mixture of pathogen(s) and the normal microbiota is present [85]. Possible culture-independent approaches that may be used to tackle the problem include metagenomic sequencing [86] and single cell genome sequencing [87]. However, these have to be tested in the clinical setting in the future before put into routine use.…”

Section: Discussionmentioning

confidence: 99%

Fighting Outbreaks with Bacterial Genomics: Case Review and Workflow Proposal

Cheung

Kwan²

2012

Public Health Genomics

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Background: Disease outbreak investigation is a key aspect of public health. Whole-genome sequencing of bacterial pathogen based on new generation high-throughput sequencing technologies has facilitated outbreak investigations recently. Whilst the approach has become more affordable and accessible to research and clinical laboratories, a system for adequate and efficient analyses of genome data in the context of bacterial outbreak investigations is missing. Methods: We performed a literature review of timely genomic investigations performed during the course of bacterial outbreaks that are based on new generation sequencing technologies. Currently available bioinformatics tools for genomic analyses are also reviewed here. Results: Genomic investigations in early stages of bacterial outbreaks have shown to provide timely information on evolutionary origin, transmission route, pathogenic potential, and resistance information of the outbreak strains and allow development of strain-specific typing methods. A systematic genomic analytical workflow is proposed here for the first time to facilitate efficient extraction of epidemiologically useful information from genome data of bacterial pathogens in future bacterial outbreak investigations. Conclusion: With the continuous reduction of genome sequencing cost and development of user-friendly analytical tools, it is expected that high-throughput genome sequencing will be applied routinely for timely genomic analysis in bacterial outbreaks in the near future.

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Single cell genome sequencing

Cited by 90 publications

References 45 publications

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

Fighting Outbreaks with Bacterial Genomics: Case Review and Workflow Proposal

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