Whole genome amplification and de novo assembly of single bacterial cells

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.

Figure 1. Sequencing coverage after WGA of individual Prochlorococcus cells.

A) Average fold-coverage within 100 bp windows was determined for SAG A from the alignment of 454-FLX read data (blue) and Illumina read data (red) to the Prochlorococcus MED4 reference genome. B) Genome recovery for individual Prochlorococcus cells, SAG A and SAG B, as a function of sequencing effort for Illumina libraries (35 nucleotide read lengths). Each datapoint represents the mean genome recovery from at least two reference-guided assemblies. Standard deviations are too small to be visible on the figure.

Figure 2. Variation in coverage depth among replicate single-cell WGA libraries.

Coverage maps generated from Illumina sequencing data (35 nucleotides read lengths) for seven replicate Prochlorococcus single-cell amplified genomes (SAGs) are overlayed. SAG A (pink), SAG B (blue), SAG C (grey), SAG D (red), SAG E (green), SAG F (orange), and SAG G (purple).

Figure 3. Impact of library normalization on coverage variation.

A) The relative abundance of 8 loci distributed across the entire genome of SAG A before and after normalization. Error bars represent one standard deviation of three separate normalization reactions. B) Coverage depth of SAG A prior to normalization. C) Fraction of genome covered at >1X and >100X in normalized and non-normalized control libraries of SAG A. Error bars representing 95% confidence intervals are obscured by symbols. D) Coverage depth of SAG A after normalization.

Figure 4. Comparison of de novo assembly results using 454-FLX and Illumina sequencing data.

A) Fraction of genome (solid bars) and number of contigs (stripped bars) assembled de novo using various sequencing approaches. The sequencing efforts on each platform were: Illumina 35 nucleotides (nt) = 455X, Illumina 71nt = 407X, Illumina 71 nt normalized = 407X, 454-FLX = 39X, and Hybrid 454 and Illumina = 446X. B) Genome recovery from normalized and un-normalized Illumina 71 nt libraries as a function of sequencing effort.