Optical mapping as a routine tool for bacterial genome sequence finishing.

Phil Latreille,Stacie Norton,Steve Forst,Zijin Du,Brad Barbazuk,Nancy Miller,Helge B Bode,Brad Goodner,Heidi Goodrich-Blair,Sophie Gaudriault,John Henkhaus,Barry S Goldman,Steven Slater,Creg Darby

doi:10.1186/1471-2164-8-321

Abstract

BackgroundIn 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.ResultsWhole 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.ConclusionOur 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.

Highlights

In sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts
A whole-genome restriction map permits finishing of the X. nematophila genome sequence Eight-fold genome sequence coverage of X. nematophila ATCC19061 (Goodrich-Blair et al, in preparation) was generated, with 26,976 reads from a 2–4 kb insert library and 41,376 reads from a 4–8 kb insert library
Using the paired clone-end sequences and syntenic comparison to the related species Photorhabdus luminescens [13], resolution of misassembles and gap closure was attempted by walking across individual clones and amplifying potentially adjacent regions using the polymerase chain reaction (PCR)

Summary

Introduction

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. BMC Genomics 2007, 8:321 http://www.biomedcentral.com/1471-2164/8/321 insect larvae that are the prey of the nematode. Both organisms reproduce in the dead larvae, the Xenorhabdus colonize the young nematodes, and the cycle repeats [11]. We produced an optical map of a second genome that we had considered finished, and identified a large sequence inversion that would have otherwise been unnoticed

Methods

Results

Discussion

Conclusion