Whole genome amplification and de novo assembly of single bacterial cells
Name
Rodrigue-2009-Whole genome amplifi.pdf
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Author(s)
Rodrigue, Sebastien
Malmstrom, Rex R.
Berlin, Aaron M.
Birren, Bruce W.
Henn, Matthew R.
Chisholm, Sallie (Penny)
Date Issued
September 2009
Journal
PLoS ONE
Publisher
Public Library of Science
Citation
Rodrigue S, Malmstrom RR, Berlin AM, Birren BW, Henn MR, et al. (2009) Whole Genome Amplification and De novo Assembly of Single Bacterial Cells. PLoS ONE 4(9): e6864. doi:10.1371/journal.pone.0006864
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Department of Civil and Environmental Engineering
Terms of Use
Creative Commons Attribution
Persistent DSpace Link
DOI of Published Version
http://dx.doi.org/10.1371/journal.pone.0006864
