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Nucleic Acids Research, 2001, Vol. 29, No. 22 e110
© 2001 Oxford University Press

General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440

Christian Weinel*, Burkhard Tümmler, Helmut Hilbert1, Karen E. Nelson2 and Claudia Kiewitz

Klinische Forschergruppe OE 6711, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany, 1QIAGEN GmbH, Max-Volmer-Str. 4, D-40724 Hilden, Germany and 2The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA

A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies. The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage. In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes. Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.

* To whom correspondence should be addressed. Tel: +49 511 532 6721; Fax: +49 511 532 6723; Email: weinel.christian{at}mh-hannover.de


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