Sequence Analysis with Artemis & Artemis Comparison Tool (ACT) South East Asian Training Course on Bioinformatics Applied to Tropical Diseases (Sponsored by UNDP/World Bank/WHO/TDR) International Centre For Genetic Engineering And Biotechnology, New Delhi, INDIA

Overview of the genome sequencing and sequence analysis. Demonstration of Artemis. Hands on guided exercise in Artemis. Demonstration of ACT. Hands on guided exercise in ACT Generating ACT comparison files Workshop Overview

Wellcome Trust Photo Library The Wellcome Trust Sanger Institute Funded by The Wellcome Trust, a registered charity. Established in 1993 to begin the Human genome project. First Draft (2000) complete (2003-4) Data release policy: All sequence data is released immediately and is freely available via the internet in order to maximise its benefit for research. ftp://ftp.sanger.ac.uk/ Wellcome Trust Photo Library

Generating the complete genome sequence

Infrastructure

Levels of automation Colony picking robots Plasmid preps robots TOTAL:140 ABI3700 ABI3730

Automated sequencing Each ABI reads 96 DNA sequences at once. The machines are run 10 times a day, 7 days a week. Throughput of 1,200 to 1, well plates per day ± 120,000 DNA samples read each day. Each day, the Sanger Institute reads 60 million base pairs. That’s equal to one of the smaller human chromosomes and many times that of an average bacterial genome.

Pathogen Sequencing Unit Bacteria: M. tuberculosis M. leprae Y. pestis S. typhi C. Diphtheriae Bordetella spp. x3 B. pseudomallei S. aureus MRSA S. aureus MSSA E. carrotovora Yeasts and Fungi: Saccharomyces cerevisiae Schizosaccharomyces pombe Aspergillus fumigatus Candida dubliniensis Candida parapsilosis Protozoa: Plasmodium falciparum X3 Plasmodium spp. X5 Leishmania spp. Trypanosoma spp. Eimeria Theileria Babesia The Pathogen Group is funded by the Beowulf Genomics Initiative to sequence the genomes of a wide range of small Eukaryotes and microbes.

Sequencing strategy and assembly

Contiguous sequence DNA pUC clone end sequence physical gap sequence gap Shotgun sequencing – strategy ‘Draft sequence’ Order of contigs? 95% coverage, 4-5x depth.

‘A genome in a day’ ‘15 in a month’ ‘High-quality draft sequence’

Contiguous sequence DNA pUC clone end sequence large clone end sequence physical gap sequence gap Shotgun sequencing – strategy Finished sequence: 100% coverage, 10x depth.

Repeats!!!

Shotgun assembly - Yersinia pestis

Primary DNA sequence Dotter BlastN BlastX Gene finders tRNA scan RepeatsPseudo-genesrRNA Genes tRNA Manual curation

Primary DNA sequence Dotter BlastN BlastX Gene finders tRNA scan RepeatsPseudo-genesrRNA Genes tRNA FastaBlastPPfamPrositePsortSignalPTMHMM Manual curation Manual curation Annotated sequence

PSU Projects Organism Annotated genome Finished genome Database entry Artemis

Sequence viewer and analysis tool –Visualization of sequence features DNA Six frame translation –Perform and view analysis Basic analysis Launch more complex analysis and searches Import and view the results of other searches

Outline of Artemis demonstration Artemis window features Open a genome sequence Changing the view Getting around –Goto Menu –Navigator –Feature Selector Basic analysis –Edit a feature –Fasta search –Show feature plots

Artemis Sliders Drop Down Menus Entry Button Line Main Sequence View Panel Magnified Sequence View Panel Feature Menu Drop Down Menus Entry Button Line Main Sequence View Panel Magnified Sequence View Panel Feature Menu

Artemis

Curating gene models in Artemis Use of multiple lines of evidence

Curating gene models in Artemis Use of FASTA evidence

EST sequencing & mapping AAAAAAAAAA CAP AAAAAAAAAA CAP TTTTTTTTT intron exon 5’UTR M stop 3’UTR EST cDNA mRNA

ESTs Curating gene models in Artemis Use of EST evidence

Curation of gene models in Artemis Mapping proteome fragments to genome

Curation and annotation in Artemis Mapping InterPro domain hits to genome

Finished sequence Gene Finder PHAT Glimmer Orpheus FASTA BLAST EST Primary gene model Annotation of pathogen genomes at the PSU (using ARTEMIS) Complete Annotation Organism-specific gene familiesFunctional classification (GO / Riley) Comparative genomics (using ACT) Refined gene model InterPro scan HMMPfam HMMSMART PRINTS PROSITE ProDom TIGRFAMs Manual curation SignalP TMHMM t-RNA scan

Gene model annotation Gene function

Top tips! Manual annotation. Use a several lines of evidence: - Run several available gene finding programs - Search programs: local (BLAST) and global (FASTA) alignments -Protein domains and motifs: Interpro (Pfam, prosite, SMART etc.) -Transmembrane / signal peptide prediction (TMHMM, SignalP) - Base your annotation on characterised proteins where possible (e.g. UNIPROT entry) - Read the literature (Pubmed entry)

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