General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440.
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.
Weinel
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Klinische Forschergruppe OE 6711, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. weinel.christian@mh-hannover.de
Tümmler
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Hilbert
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Nelson
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Kiewitz
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D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D004706:Endodeoxyribonucleases / Q000378:metabolism; D016680:Genome, Bacterial; D016958:Pseudomonas putida / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics; D015183:Restriction Mapping / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D012407:rRNA Operon / Q000235:genetics
D004269:DNA, Bacterial; D012335:RNA, Ribosomal; D004706:Endodeoxyribonucleases; C071132:endodeoxyribonuclease I-CeuI; C074767:endodeoxyribonuclease SwaI; D015252:Deoxyribonucleases, Type II Site-Specific