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A generic and modular platform for automated sequence processing and annotation

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. Sequence processing and annotation. Analyzing and processing sequencing reads is a tedious and error-prone job

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A generic and modular platform for automated sequence processing and annotation

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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. 2 Solution: creating an environment to build pipelines • 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) Requirements: 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 • 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 Characteristics: AG-ICB-USP

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  15. 2 Sequence processing pipelineThe Eimeria ORESTES project Mitochondrial sequence filtering Cross_Match Input chromatogram files Plastid sequence filtering Base calling and quality assignment Cross_Match Phred Ribosomal sequence filtering Cross_Match Primer screening and masking 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 Human sequence filtering Blast Size filtering Filter-size Assembly CAP3 AG-ICB-USP

  16. 2 Sequence processing and grahical report AG-ICB-USP

  17. 2 How to get EGene Internet site: http://www.coccidia.icb.usp.br/egene - EGene is distributed under the GNU General Public License - EGene is Open Source AG-ICB-USP

  18. 2 How to get EGene Internet site: http://www.coccidia.icb.usp.br/egene - EGene is distributed under the GNU General Public License - EGene is Open Source AG-ICB-USP

  19. 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

  20. 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

  21. 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

  22. 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

  23. 2 Feature table format • Flatfile format • Format definition available at http://www.ncbi.nlm.nih.gov/projects/collab/FT/ • Covers DDBJ/EMBL/GenBank • Defines all accepted annotation terms and hierarchy AG-ICB-USP

  24. 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

  25. 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

  26. 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 • Web site generation: HTML/PHP AG-ICB-USP

  27. 2 EGene generates annotation files that can be inspected using regular editors (Artemis, Apollo, etc.) AG-ICB-USP

  28. 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

  29. 2 EGene performs GO term mapping and constructs web pages for inspection AG-ICB-USP

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