Optical mapping as a routine tool for bacterial genome sequence finishing

Abstract

Background: In sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila.

Results: Whole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished.

Conclusion: Our experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly.

Figure 1

Alignments between the whole-genome optical maps and the in silico genome sequence assemblies at various stages of the project. Green regions indicate perfect alignment, white regions indicate no alignment, red regions indicate sequence that is present on at least two contigs, and yellow regions indicate inversions. Lines between maps indicate the position of identical sequences on the two maps, and can be used to visually identify misassemblies and inversions. Panel A: An early comparison of an optical map derived from EagI digestion of the X. nematophila genome to the assembled contigs generated by traditional sequencing technologies. All contigs could be ordered for gap closure. In addition, the optical map indicated an overlooked misassembly. Panel B: The finishing strategy, including gap closure and misassembly resolution, was simplified using the optical map as an assembly model. The X. nematophila optical map derived from an AflIII digestion of the chromosome is presented as a single contig in the center. The sequenced genome contains nine contigs that have a corresponding match to the optical map. The X. nematophila plasmid is 158 Kb and is too small to be identified using the current optical map technology. Nonetheless, small sections of the plasmid can be identified as regions that do not have corresponding optical map locations (white in figure). Panel C: Comparison of the final assembly of the X. nematophila genome (bottom) to the optical map (top) for the EagI digest. The non-aligned contig represents the plasmid, which was generated by traditional sequencing technologies. Panel D: Comparison of the finished sequence of Xenorhabdus bovienii to the EagI optical map revealed a large inverted region of the genome. The red regions indicate regions of repeats within the genome that cannot be resolved by optical mapping. These regions were resolved using traditional sequencing methods. The sequenced genome was easily re-oriented to correct the assembly.