Whole genome amplification and de novo assembly of single bacterial cells.
Sébastien Rodrigue,Rex R. Malmstrom,Aaron M. Berlin,Bruce W. Birren,Matthew R. Henn,Sallie W. Chisholm +5 more
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TLDR
A pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions is described and a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification is developed.Abstract:
Background: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells. Methodology/Principal Findings: We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. Conclusions/Significance: The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.read more
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Journal ArticleDOI
SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing
Anton Bankevich,Sergey Nurk,Dmitry Antipov,Alexey Gurevich,Mikhail Dvorkin,Alexander S. Kulikov,Valery M. Lesin,Sergey I. Nikolenko,Son Pham,Andrey D. Prjibelski,Alexey V. Pyshkin,Alexander Sirotkin,Nikolay Vyahhi,Glenn Tesler,Max A. Alekseyev,Pavel A. Pevzner +15 more
TL;DR: SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies.
SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)
TL;DR: SPAdes as mentioned in this paper is a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler and on popular assemblers Velvet and SoapDeNovo (for multicell data).
Journal ArticleDOI
Idba-ud
TL;DR: Comparison of the performances of IDBA-UD and existing assemblers (Velvet, Velvet-SC, SOAPdenovo and Meta-IDBA) for different datasets, shows that IDba-UD can reconstruct longer contigs with higher accuracy.
Journal ArticleDOI
Isothermal Amplification of Nucleic Acids
TL;DR: This review provides a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades including applications in bioanalysis, diagnostics, nanotechnology, materials science, and device integration.
Journal ArticleDOI
Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes
Mads Albertsen,Philip Hugenholtz,Adam Skarshewski,Kåre Lehmann Nielsen,Gene W. Tyson,Per Halkjær Nielsen +5 more
TL;DR: Reanalysis of published metagenomes reveals that differential coverage binning facilitates recovery of more complete and higher fidelity genome bins than other currently used methods, which are primarily based on sequence composition.
References
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TL;DR: An approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost is reported, effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.