Whole genome amplification and de novo assembly of single bacterial cells.
Rodrigue S, Malmstrom RR, Berlin AM, Birren BW, Henn MR, Chisholm SW
PLoS One. 2009 Sep 2;4(9):e6864.
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
Additional Raw Data