Whole Genome Amplification and De Novo Assembly of Single Bacterial Cells

PLoS One. 2009 Sep 2;4(9):e6864. doi: 10.1371/journal.pone.0006864.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteria / genetics
  • Cell Separation
  • Chromosome Mapping / methods
  • Flow Cytometry
  • Gene Library
  • Genes, Bacterial / genetics*
  • Genetic Variation
  • Genome, Bacterial*
  • Genomics / methods
  • Humans
  • Models, Biological
  • Nucleic Acid Amplification Techniques / methods*
  • Prochlorococcus / genetics
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods*