An improved genome of the model marine alga Ostreococcus tauri unfolds by assessing Illumina de novo assemblies

Abstract

Background: Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture.

Results: The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene.

Conclusion: High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture.

Figure 1

Illumina DNAseq and RNAseq aligned against Ostreococcus tauri reference genome sequence. Colored numbered lines represent the 20 chromosomes of Ostreococcus tauri. The contiguity of the de novo assembly along the chromosomes ranges from 0 (white) to 28 scaffolds per 30 kb window (red). The inner blue track is the DNAseq coverage (from 0 to 582 reads per bp). The inner purple track is the RNAseq coverage averaged across 10 kb windows (from 0 to 1947 reads per bp). Figure generated with the RCircos software [51].

Figure 2

Saturation curve of coverage along the GenBank reference genome sequence. BWA alignment of 41 M Illumina paired-end reads subsets representing different sequencing depth (black line) and after NUCmer alignment of de novo scaffolds produced by a Velvet de novo assembly of these same paired-end reads subset (grey line).

Figure 3

Localization of the substitutions between 2001 and 2009 within two genes. A: ostta06g00130 (Ot06g00160), B: gene organization of ostta12g00065 (Ot12g00160), C: Transmembrane organization of the two encoded proteins, left : Arabidopsis glutamate-like receptors homologous to Ot12g00160 from Lam et al. [58], right : TMHMM prediction for Ot06g00160.