PMC:2952849 / 6331-9256 JSONTXT

{"project":"2_test","denotations":[{"id":"20478826-2231712-76650475","span":{"begin":613,"end":615},"obj":"2231712"},{"id":"20478826-12783628-76650476","span":{"begin":888,"end":890},"obj":"12783628"},{"id":"20478826-11410670-76650477","span":{"begin":904,"end":906},"obj":"11410670"},{"id":"20478826-14988122-76650478","span":{"begin":921,"end":923},"obj":"14988122"},{"id":"20478826-12626712-76650479","span":{"begin":1701,"end":1703},"obj":"12626712"},{"id":"20478826-12824352-76650480","span":{"begin":2420,"end":2422},"obj":"12824352"}],"text":"Whole-genome sequencing, annotation and bioinformatic procedures\nThe genome of H. pylori strain P12 was sequenced by a whole-genome shotgun method. Bacteria were subjected to minimal passage before DNA was prepared by the cesium chloride method. Two shotgun libraries of 2 kb and of 5 kb were constructed in pSMART-hcKan (Lucigen). High-throughput sequencing of the libraries was performed using ABI 3730 technology to a 5-fold theoretical coverage. Raw sequence data were assembled with the SeqMan program of the LaserGene software package (DNASTAR). Metacontigs obtained by SeqMan were arranged based on BLAST (18) alignments to the genomic sequences of strains 26695, HPAG1 and J99. Gaps were filled by primer walking on shotgun library plasmids or by sequencing PCR fragments. Coding sequences were predicted based on homology to 26695, HPAG1 and J99 proteins as well as EasyGene1.0 (19), GeneMarkS (20) and Reganor (21) predictions. Open reading frames were assumed to be coding if they were predicted by at least two of the three prediction-tools or if their translated sequences showed significant similarities to 26695, HPAG1 or J99 proteins (using tblastn; thresholds: bit-score \u003e100, e-value \u003c10−10, identity \u003e30%, proportion of aligned ORF-sequence \u003e66%) and were at least 40 amino acids long. Start codons (ATG, GTG or TTG) were chosen using majority vote (tool predictions and homology approach); if non-unique, the most upstream start codon was chosen. Gene predictions were partly manually curated. Assignment of protein functions was based on the annotations of H. pylori strain HPAG1, H. acinonychis strain Sheeba as well as PyloriGene annotations of H. pylori strains 26695 and J99 (22), BLASTP searches versus nr- and cdd-databases (thresholds: E-value \u003c10−20, identity \u003e35%) as well as manual curation. Groups of orthologous genes were derived based on homology and synteny, allowing identification of orthologs despite possibly low sequence homology, insertion/deletion or recombination events, frameshifts, or premature stops. An orthologous group contains all genes or gene fragments presumably originating from a single ancestor gene, although this ancestor gene may be split in all organisms considered. Dendrograms of VirB4, VirB9 and VirB10 homologs of H. pylori 26695, HPAG1, J99, Shi470, G27 and P12, H. acinonychis Sheeba, and C. jejuni 81-176 (pTet and pVir) were created using ClustalW2 (23). Pairwise distances of homologs were obtained from these dendrograms, and distances between whole type IV secretion systems were calculated as averages of gene-to-gene distances. Type IV systems were clustered using Neighbor Joining. A phylogenetic tree of H. pylori and H. acinonychis strains based on nucleotide similarity of the core genome was calculated as a consensus tree of 1117 independent phylogenetic trees for each orthologous group of genes within the core genome of the strains examined."}