PMC:3091627 / 29907-32905 JSONTXT

{"project":"2_test","denotations":[{"id":"20537153-9521922-10781753","span":{"begin":875,"end":877},"obj":"9521922"},{"id":"20537153-9521923-10781754","span":{"begin":1121,"end":1123},"obj":"9521923"},{"id":"20537153-11282977-10781755","span":{"begin":1226,"end":1228},"obj":"11282977"},{"id":"20537153-12824403-10781756","span":{"begin":2424,"end":2426},"obj":"12824403"},{"id":"20537153-16407324-10781757","span":{"begin":2548,"end":2550},"obj":"16407324"}],"text":"Sequencing and annotation of the B38 genome\nGenomic DNA was randomly sheared by nebulization (HydroShear, GeneMachines) and the ends were enzymatically repaired. SmaI fragments (1.5-4 kb) were inserted into plasmid vector pBAM3/SmaI (derived from pBluescript KS and constructed by R. Heilig). Large (35-45 kb) DNA fragments generated from partial BamHI-restriction were inserted into the cosmid vector pHC79/BamHI.\nPlasmid DNA was prepared with the TempliPhi DNA sequencing template amplification kit (GE Healthcare-Bio-Sciences). Cosmid DNA was purified with the Montage BAC Miniprep96 Kit (Millipore). Sequencing reactions were performed from both ends of DNA templates using ABI PRISM BigDye Terminator cycle sequencing ready reactions kits and were run on a 3700 or a 3730 xl Genetic Analyzer (Applied Biosystems).\nSequence data base calling was carried out using Phred [40]. Sequences not meeting our production quality criteria (at least 100 bases called with a quality over 20) were discarded. Sequences were screened against plasmid vector and E. coli sequences. The traces were assembled using Phrap and Consed [41]. Whole genome shotgun sequencing was performed to ensure approximately 11-fold coverage. Autofinish [42] was used to design primers for improving regions of low quality sequence and for primer walking along templates spanning the gaps between contigs. Several strategies were used to orientate contigs and to enable directed PCR-based approaches to span the gaps between contigs. These strategies included linking isolates and a Blast-based approach, which identified contigs with hits to the H. pylori strain 26695 genome. Various combined PCR techniques were used to amplify genomic or cosmid DNA, to close the gaps between the final contigs. Outward-directed primers were designed for each of the contig ends; the primer sequences were subsequently checked and confirmed to be unique to the genome. This combined PCR process required approximately 200 PCR reactions pairing each of the primers. In addition, two cosmid isolates containing a rDNA operon copy each, were completely sequenced by sub-cloning into a pSMART-LC vector (Lucigen Corp.). The error rate was less than 1 error per 10,000 bp in the final assembly. The complete genome sequence was obtained from 40 153 sequences, resulting in 14-fold coverage.\nAMIGene software was used to predict which CDSs were likely to encode proteins [43]. The set of predicted genes underwent automatic functional annotation using the set of tools listed in Vallenet et al. [28]. All these data (syntactic and functional annotations, results of comparative analysis) are stored in a relational database, called PyloriScope. Manual validation of the automatic annotation was performed using the MaGe (Magnifying Genomes, http://www.genoscope.cns.fr) web-based interface, which allows graphic visualization of the annotations enhanced by the synchronized representation of synteny groups in other genomes chosen for comparison."}