PMC:1069646 / 47242-52140 JSONTXT

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Because of the difficulties in obtaining purified Wolbachia DNA from the B. malayi host, bacterial artificial chromosome (BAC) libraries were created [114]. From these libraries, a minimum tiling path of 21 Wolbachia BACs was created and used for subcloning into plasmid vectors for genomic sequencing. This ordered BAC approach was useful in the assembly phase of the project because of the highly repetitive nature of this genome.\nFor plasmid library generation, equal amounts of BAC DNAs were pooled and 50 μg of DNA from the pool was sheared into 2.0–3.0 kb fragments (HydroShear device, GeneMachines, Genomic Solutions, Ann Arbor, Michigan, United States). Sheared DNA was purified from a 0.7% agarose gel, blunted, and cloned into cleaved, dephosphorylated plasmid vectors. Libraries were generated containing DNA from 1 to 9 BACs.\nPlasmid DNA was isolated by a modified alkaline lysis protocol. Sequencing reactions were performed at Integrated Genomics (Chicago, Illinois, United States) using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Little Chalfont, United Kingdom). Unincorporated dye was removed by isopropanol precipitation as recommended by the manufacturer. Samples were run on MegaBace 1000 (Amersham Biosciences) sequencers; 87% of plasmid sequencing reactions were successful. The genome was sequenced to an average coverage of 10.7X and at 2X minimum coverage (at least once in each direction) and assembled.\nThe sequence was assembled into contigs by using PHRED–PHRAP–CONSED [165,166,167], and gaps were initially closed by primer walking (1,766 reactions). Regions considered to be potential frame shifts or sequencing errors after the first round of annotation were resequenced from direct genomic PCR products. The completed sequence was used to identify homologous sequences in the independent ongoing B. malayi sequence project (TIGR parasites genome database: http://www.tigr.org/tdb/e2k1/bma1/ [126]). The sequence of one BAC had been previously determined [163]. The final assembly was in full agreement with the BAC physical map [114].\nIntegrated Genomics ERGO software [168] and other software programs [169] were used for ORF calling, gene identification, and feature recognition. Computational analysis of the genome sequence was performed as previously described. Briefly, the tRNA genes were identified using the tRNA-SCAN program [170], and the rRNA genes were identified using the BLASTN program [171]. For the identification of the protein-coding genes, the genome sequence was conceptually translated in six frames to generate potential protein products of ORFs longer than 100 codons. These potential protein sequences were compared to the database of proteins from the COG database using COGNITOR [172].\nAfter manual verification of the COG assignments, the validated COG members from wBm were called as protein-coding genes. The COG assignment procedure was repeated with ORFs of greater than 60 codons from the intergenic regions. Additionally, the potential protein sequences were compared to the nonredundant protein sequence database using the BLASTP program [171] and to a six-frame translation of unfinished microbial genomes using the TBLASTN program [171], and those sequences that produced hits with E (expectation) values less than 0.01 were added to the protein set after an examination of the alignments. Finally, protein-coding regions were predicted using the GeneMarkS program [173]. After manual refinement, the genes predicted with these methods in the regions between evolutionarily conserved genes were added to produce the final protein set. Protein function prediction was based primarily on the COG assignments. In addition, searches for conserved domains were performed using the Conserved Domain Database (CDD) search option of BLAST (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and the SMART system [174], and in-depth, iterative database searches were performed using thePSI-BLAST program [175]. The KEGG database [176] (http://www.genome.ad.jp/kegg/metabolism.html) and the Integrated Genomics ERGO database pathway collection [168] were used, in addition to the COGs, for the reconstruction of metabolic pathways. Paralogous protein families were identified by single-linkage clustering after comparing the predicted protein set to itself using the BLASTP program [171]. Signal peptides in proteins were predicted using the SignalP program [177], and transmembrane helices were predicted using the MEMSAT program [178]. Gene orders in bacterial genomes were compared using the Lamarck program [179].\nTwo closely related genome sequences were completed and published since the above comparative analysis was undertaken [180,181].\n"}