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Fig. 2 Genes (or segments) from each genome are compared with a reference gene, as shown in the left panel; a pairwise comparison is made using one of the BLAST algorithms. On the right is shown the ‘‘remapping’’, or the representation of each of the BLAST runs on the left, mapped onto the chromosomal sequence. Note that gaps in the reference gene (grey) are not included in the colored maps of the atlas. genome and custom data and DNA properties are collected, an XML configuration file is composed which contains all these data and the layout of the atlas. This file is then sent to the GeneWiz 7 software which produces a PostScript document, it then is base64 encoded to allow transport via XML. This part of the process takes place on the server and requires no user-interaction. An example atlas of a plasmid is shown in Fig. 3, and will be discussed in more detail below. Web services implementation A WSDL (web services description language) file is written which describes the operations (runAtlas, pollQueue, fetch- AtlasResult) and the input requirements for them. The file can be downloaded. All input/output objects are defined in a separated XSD file (XML schema definition) within the WSDL file, which comprises information and type restrictions applicable in the request. This serves as documentation of the objects as well as a way to validate a request before it is submitted. Unfortunately, the validation supports only Perl modules for now that is not optimal yet, whereas this option is well implemented in tools like soapUI (http://www.soapui.org/). It should be stressed that users should, until better validation support can be implemented, be careful to correctly format the input parameters before sending the request. Fig. 3 BLASTatlas of pE88—a small plasmid of Clostridium tetani strain E88, GenBank accession number AF528097. DNA parameters percent AT, GC skew, global direct repeats, and global inverted repeats are included in the inner most lanes. BLAST lanes of all complete genome sequences of the Clostridium genomes (see Table 1), including plasmids are included in the outer most lanes. As examples of custom lanes, the free energy (G, blue kcal mol 1 ) and the probability (P, red) measures of stress induced DNA duplex destabilization (SIDD) sites are included in the lanes between the DNA properties and the BLAST lanes. 23 SIDD calculations were obtained from the SIDDbase WebService (http://www.cbs.dtu.dk/ws/SIDDbase). The request XML used to construct this plot can be downloaded from the example section of the service homepage, http://www.cbs.dtu.dk/ws/BLASTatlas. As expected, there is full homology of all coding regions between the plasmids and all replicons of C. tetani E88 (black lane just outside of the annotations); however there appears to be limited conservation of these pE88 genes throughout the genomes for other Clostridium strains. 366 | Mol. BioSyst., 2008, 4, 363–371 This journal is c The Royal Society of Chemistry 2008
Table 2 A list of all strains and their accession numbers used in this comparison. Each row represents the NCBI Entrez sequencing project. The number of base pairs and protein coding genes are those derived as the sum within each project. C. botulinum str. F Langeland is that used as reference of the comparison Species Segments Size Proteins C. acetobutylicum ATCC 824 14 Entrez Project 77: Chromosome: AE001437, Plasmid pSOL1: AE001438 4.132.880 3.848 C. beijerinckii NCIMB 8052 (unpublished) Entrez Project 12637: Chromosome: CP000721 6.000.632 5.020 C. botulinum A str. ATCC 19397 (unpublished) Entrez Project 19517: Chromosome: CP000726 3.863.450 3.552 C. botulinum A str. ATCC 3502 6 Entrez Project 193: Chromosome: AM412317, Plasmid pBOT3502: AM412318 3.903.260 3.671 C. botulinum A str. Hall (unpublished) Entrez Project 19521: Chromosome: CP000727 3.760.560 3.407 C. botulinum F str. (unpublished) Entrez Project 19519: Chromosome: CP000728, Plasmid pCLI: CP000729 4.012.918 3.659 C. difficile 630 15 Entrez Project 78: Chromosome: AM180355, Plasmid pCD630: AM180356 4.298.133 3.787 C. kluyveri DSM 555 (unpublished) Entrez Project 19065: Chromosome: CP000673, Plasmid pCKL555A: CP000674 4.023.800 3.913 C. novyi NT 16 Entrez Project 16820: Chromosome: CP000382 2.547.720 2.325 C. perfringens ATCC 13124 25 Entrez Project 304: Chromosome: CP000246 3.256.683 2.876 C. perfringens SM101 17 Entrez Project 12521: Chromosome: CP000312, Plasmid 1: CP000313, Plasmid 2: CP000314, Viral segment phage phiSM101: CP000315 2.960.088 2.631 C. perfringens str. 13 18 Entrez Project 79: Chromosome: BA000016, Plasmid pCP13: AP003515, 3.085.740 2.723 C. tetani E88 19 Entrez Project 81: Chromosome: AE015927, Plasmid pE88: AF528097 2.873.333 2.432 C. thermocellum ATCC 27405 (unpublished) Entrez Project 314: Chromosome: CP000568 3.843.301 3.191 Clostridium phage 20 Phage c-st: AP008983 185.683 198 Web services workflow A workflow was written in Perl (v5.8.7), employing SOAP:Lite (v0.69) which reads the FASTA files of the database strains listed in Table 3 and produces a BLASTatlas using the C. botulinum strain F Langeland as reference. The script uses the online web service (see Fig. 4). The BLASTatlas figure produced by this workflow is seen in Fig. 5. Results Fig. 3 represents a BLASTatlas for plasmid pE88 from Clostridium tetani strain Fig. 4 Workflow description: a Perl script was written for handling the assembly of the SOAP envelope and contacting various other web services operations: (A) obtaining genomes sequence: using the getSeq operation of the GenomeAtlas Web Services (v.3.3), the genome sequence of the reference genome is obtained as one continuous string. (B) Obtaining atlas annotations: annotated CDS, rRNA, and tRNA features of the GenBank record of the reference genome using the getFeatures operation—these are the features which will be printed in a separate lane on the atlas. (C) Obtaining ORF annotations of the reference genome: again, using the getFeatures operation, all codon sequences and their translations are obtained. (D) Obtain databases: read FASTA files containing proteins and ORFs of the database genomes to be added as lanes. The output of A–F are assembled into a single SOAP request, including configurations of the atlas. (E) Polling the queue: once the job has been submitted, a 32 character hex string is returned for identifying the job, which can be used by operation pollQueue to see the status of the job. (F + G) Obtaining result: once a status ‘‘FINISHED’’ is obtained from pollQueue, the job id can submitted to fetchResult and the resulting PostScript image is returned. E88. The homology for genes in the plasmid to other sequenced genomes is shown in the circles, additional ‘‘custom lanes’’ represent chromosomal regions predicted to open under superhelical stress. The chromosomal location of the genes encoding colT and tetR are labelled in the figure. Notice that these two proteins contain regions of homology that are found in most of the Clostridium proteomes searched. Since the C. tetani plasmid is included in the genome sequence (black circle in the figure), all the genes are found in this genome (solid black), and most of the other Clostridium proteomes contain some weak homology but in general lack most of the plasmidencoded genes. Thus, this is a quick overview of gene conservation of a plasmid compared to many sequenced genomes of the same genera. To demonstrate this for an entire bacterial genome (which is millions of bp in size, compared to a small/B75 000 bp plasmid, shown in Fig. 3), we have used the genome sequence of C. botulinum strain F Langeland, the largest of the C. botulinum genomes, to build a protein BLASTatlas of all publicly available fully sequenced Clostridia genomes, including all chromosomes, plasmids and phages (see Fig. 5). Each lane of the atlas corresponds to a sequencing project that contains the main chromosome plus any This journal is c The Royal Society of Chemistry 2008 Mol. BioSyst., 2008, 4, 363–371 | 367
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Peter Fischer Hallin | 2009 Peter F
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Preface This Ph.D. thesis is writte
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Gap F 2M 2.5M Gap E 875k 750k 625k
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Table 2 A selection of genes locate
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Open Access This article is distrib
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1 Comparative Genomics 2.10 Paper V
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4314 74 Tools Abstract: Of the plet
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4316 74 Tools Size distribution of
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4318 74 Tools Genome atlas Intrinsi
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4320 74 Tools Genome atlas Intrinsi
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4322 74 Tools for Comparison of Bac
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Chapter 3 rRNA operons and promoter
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tuB murI Fis III Fis II Fis I UP -3
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Using HMMs also simplifies the use
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of the annotation. Some of the majo
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where match states stop around 10 c
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synthesis in flow cells to simultan
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Read absence. A boolean where ‘on
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Hallin, et al. Figure 4 | The dataf
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Genome homology: Comparing multiple
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ing platform-‐independent Java
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34. Wang H, Noordewier M, Benham CJ
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Chapter 4 Web Services and Interope
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Web Services and Interoperability i
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Chapter 5 Conclusion and perspectiv
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Appendix A Appendix: Workshops, tea
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Appendix B Appendix: Ph.D. study pl
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Danmarks Tekniske Universitet AFI,
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Danmarks Tekniske Universitet AFI,
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Appendix C Appendix: Courses C.1 Gl
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D.2 Sample output from queryGenomes
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Appendix: Software 25 [ ] A l t e r
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BIBLIOGRAPHY J. Rogers, P. F. Stadl
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BIBLIOGRAPHY Q. Jin, Z. Yuan, J. Xu
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BIBLIOGRAPHY Velicer, F.-J. Vorholt
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