A generic and modular platform for automated sequence processing and annotation. Arthur Gruber
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1 2 A generic and modular platform for automated sequence processing and annotation Arthur Gruber Instituto de Ciências Biomédicas Universidade de São Paulo AG-ICB-USP
2 2 Sequence processing and annotation Analyzing and processing sequencing reads is a tedious and error-prone job Multistep process All sequences are submitted to the same processing steps Sequences processed by a given step are the input for the next one Require different programs Integrated system PIPELINE AG-ICB-USP
3 2 Problem: how to build pipelines Creating scripts for new pipelines involves good programming knowledge Once created, most pipelines are difficult to change and customize Many programs must be used Phred, Cross_match, Phrap, CAP3, Blast, HMMer, InterproScan, TMHMM, etc. AG-ICB-USP
4 2 Problem: how to build pipelines Each program needs a specific environment to work (e.g. directories with specific names) Each program produces output in different ways and formats Integrating programs is a hard task AG-ICB-USP
5 Solution: creating an environment to build pipelines 2 Requirements :Abstract the environment of each program Abstract output format Easily specify coupling of different programs Document how the pipe was built Easy to inspect and monitor Easy to store (e.g. in a database) AG-ICB-USP
6 2 EGene Aims and characteristics: To develop a simple to use and configure platform for pipeline construction Big sequencing centers already have sophisticated pipelines, but many are not published and/or publicly available They are too complex for the small-/mid-sized labs Platform should be generic Useful for any sequencing project Platform should provide components for the most common tasks New components should be easy to develop AG-ICB-USP
7 2 EGene: a generic platform for pipeline construction Characteristic s: Written in Perl language Modular Easy to build specific components to interact with third-party programs EGene components can be integrated to fulfill user-specific needs CoEd a graphical configuration editor written in Java user-friendly interface AG-ICB-USP
8 AG-ICB-USP AG-ICB-USP
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15 2 Sequence processing pipeline The Eimeria ORESTES project Input chromatogram files Base calling and quality assignment Phred Primer screening and masking Cross_Match Mitochondrial sequence filtering Cross_Match Plastid sequence filtering Cross_Match Ribosomal sequence filtering Cross_Match Repetitive sequence filtering Cross_Match Vector masking and screening Cross_Match Bacterial sequence filtering Blast Quality filtering Filter-quality.pl Chicken sequence filtering Blast End trimming Trim-ends.pl Size filtering Filter-size Human sequence filtering Blast Assembly CAP3 AG-ICB-USP
16 2 Sequence processing and grahical report AG-ICB-USP
17 2 How to get EGene Internet site: - EGene is distributed under the GNU General Public License - EGene is Open Source AG-ICB-USP
18 2 How to get EGene Internet site: - EGene is distributed under the GNU General Public License - EGene is Open Source AG-ICB-USP
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20 2 Recent developments Incorporation of forks Enhancement of the data model incorporation of annotation evidences Development of annotation components Evidence-based annotation AG-ICB-USP
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24 2 Genome annotation Annotation is the process of adding information to DNA sequence. The information usually has a DNA coordinate. Features could be repeats, genes, promoters, protein domains, etc. Features can be cross-referenced to other databases (e.g. Pfam/Pubmed) AG-ICB-USP
25 2 Genome annotation Annotation is the process of adding information to DNA sequence. The information usually has a DNA coordinate. Features could be repeats, genes, promoters, protein domains, etc. Features can be cross-referenced to other databases (e.g. Pfam/Pubmed) AG-ICB-USP
26 2 Annotation file A typical annotation file contains: A header with: Information about the sequence Organism Authors References Comments A feature table containing Sequence features and co-ordinates AG-ICB-USP
27 2 Feature table format Flatfile format Format definition available at Covers DDBJ/EMBL/GenBank Defines all accepted annotation terms and hierarchy AG-ICB-USP
28 2 Incorporating annotation EGene s data model was enriched to incorporate annotation information into the representation of the sequences All collected data is converted into a proprietary XML format The XML can be easily converted into different annotation formats: Feature Table, GFF3, etc. We provide some converters and new ones can be easily implemented AG-ICB-USP
29 2 Annotation components A comprehensive set of annotation components has been implemented: ORF finding and translation Tandem repeats finding: TRF, String, mreps trna finding: trnascan-se Gene Prediction: Genscan, GlimmerM, GlimmerHMM, Twinscan, Phat, ESTscan, SNAP Motif finding: HMMer x Pfam, RPS-BLAST, InterproScan Similarity search: BLAST EST mapping: Sim4, Exonerate AG-ICB-USP
30 2 Annotation components A comprehensive set of annotation components has been implemented: Transmembrane domain finding: TMHMM, Phobius Signal peptide: SignalP, Phobius GPI anchor: DGPI GO mapping and quantification Orthology assignment and quantification: COG/KOG Pathway mapping: KEGG Annotation visualization with GBrowse: web inspection Annotation report generation: feature table, GFF3 AG-ICB-USP
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34 2 EGene generates annotation files that can be inspected using regular editors (Artemis, Apollo, etc.) AG-ICB-USP
35 2 EGene s annotation EGene can generate annotation in different formats: XML local use, easy to feed a database management system Feature table Convenient for manual curation on Artemis Ready for submission to public databases GFF3 Current annotation interchange format Manual curation/visualization on Artemis, Apollo and GMOD Genome Browser Compliant with Sequence Ontology terms AG-ICB-USP
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40 2 EGene performs GO term mapping and constructs web pages for inspection AG-ICB-USP
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61 2 EGene performs an integrated and quantitative orthology analysis (COG/KOG) and constructs web pages AG-ICB-USP
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82 2 EGene automatically constructs a full web site for evidence inspection AG-ICB-USP
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96 2 Current developments Full integration with management system a database Automated task distribution management across multiple processing nodes Development of a graphical interface for evidence inspection and manual curation Intelligent annotation use of probalistic methods to evaluate evidence and designate protein functions AG-ICB-USP
97 2 Ideal for small- and mid-sized laboratories Genome and EST sequencing projects Conceived for Biologists Why use EGene2? Does not require programming skills Generic tool for any sequencing/annotation project customized for specific user s requirements Very easy to implement new components Multiplatform - MacOS, UNIX, Linux, etc. Well documented HOWTOs, tutorials, example datasets available Easy configuration CoEd - Application with a GUI for pipeline construction Generic pipeline templates provided AG-ICB-USP
98 2 Research team Prof. Alan M. Durham IME-USP Annotation Milene Ferro ICB-USP Ricardo Yamamoto Abe IME-USP Luiz Thiberio Rangel ICB-USP Sequence pre-processing André Yoshiaki Kashiwabara - IME-USP Fernando Tadashi G. Matsunaga - ICB-USP Paulo Henrique Ahagon - ICB-USP Leonardo Varuzza - ICB-USP AG-ICB-USP
99 2 Financial Support FAPESP - São Paulo State Science Foundation CNPq - National Research Council AG-ICB-USP
100 Thanks for your attention AG-ICB-USP
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