De Novo Sequencing and Assembly of the Whole Genome of Novosphingobium sp. Strain PP1Y

Abstract

Novosphingobium sp. strain PP1Y is a marine bacterium specifically adapted to use fuels as an energy source. We sequenced and assembled its entire genome using the Roche 454 genome sequencer system, which led to the identification of two plasmids and one megaplasmid, besides a 3.9-Mb circular chromosome.

GENOME ANNOUNCEMENT

Novosphingobium sp. strain PP1Y, isolated from seawater from the harbor of Pozzuoli (Naples, Italy), is able to grow on a wide range of aromatic compounds, including mono-, bi-, tri-, and tetracyclic aromatic hydrocarbons and heterocyclic compounds (4). These peculiar features suggest that PP1Y has adapted to grow efficiently at the water/fuel oil interface using the aromatic fraction of the fuel oils as a source of carbon and energy (4). The whole PP1Y genome sequence could provide clues about the metabolism of this species and about the possibility of manipulating it for bioremediation purposes.

The so-called next-generation massively parallel DNA sequencing technologies have proved to be efficient tools for the sequencing of microbial genomes (1, 2, 5). We performed high-throughput de novo sequencing and assembly of the whole Novosphingobium genome using the Roche 454 genome sequencer 20 (GS20) system (3). The genomic DNA was randomly sheared into small fragments, purified, and assessed for quality. The library obtained was hybridized on the surfaces of microscopic beads, clonally PCR amplified within the droplets of a water-in-oil emulsion (emPCR), and bidirectionally sequenced using a pyrosequencing protocol. The resulting sequencing reads were analyzed and processed into contigs generating a consensus sequence. PCR amplifications were performed to fill gaps generated by contig assembly. Four sequencing runs were performed, which yielded more than 120 Mb, being equivalent to 1,224,396 sequencing reads. Because the PP1Y genome is about 5.3 Mb, we obtained a coverage of 22.6×. The average read length was 101.8 bp, and the average GC content was 61.7%. The vast majority (98.8%) of the sequencing reads were used for the assembly, which resulted in 286 contigs, 108 of which were larger than 500 bp. The largest contig was 368,742 bp. We used two PCR strategies to complete the assembly. The results were assembled with the GS20 data so that the entire genome sequence was obtained.

Subsequently, a single GS-FLX-titanium run was done, and the resulting longer reads were assembled with the GS20 reads, which showed there was no misassembly. Thus, the final coverage was higher than 88×.

The PP1Y genome is comprised of a single 3.9-Mb circular chromosome and of 3 plasmids, one megaplasmid (Mpl; 1.16 Mb), one large plasmid (Lpl; 0.19 Mb), and one small plasmid (Spl; 0.05 Mb). Notably, this is the first time that a megaplasmid of such size has been identified in a sphingomonad. Our results will help to clarify the molecular basis of the unusual features of this strain and to engineer strains with enhanced cultural and bioremediational abilities.

Nucleotide sequence accession numbers.

The whole-genome sequence obtained in this study has been deposited in the EMBL database under the following accession numbers: FR856862 (chromosome), FR856861 (Mpl), FR856860 (Lpl), and FR856859 (Spl).