Sequence Analysis with Artemis & Artemis Comparison Tool (ACT) South East Asian Training Course on Bioinformatics Applied to Tropical Diseases - 2005 (Sponsored.

Sequence Analysis with Artemis &

Artemis Comparison Tool (ACT)South East Asian Training Course on

Bioinformatics Applied to Tropical Diseases - 2005

(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.

http://www.sanger.ac.ukftp://ftp.sanger.ac.uk/

Wellcome Trust Photo Library

Generating the complete genome sequence

Infrastructure

Levels of automation

Colony pickingrobots

Plasmid prepsrobots

TOTAL:140

ABI3700

ABI3730

Automated sequencing

Each ABI reads 96 DNAsequences at once.

The machines are run10 times a day,7 days a week.

Throughput of 1,200 to 1,300 96-well plates per day ± 120,000 DNA samples read each day.

Each day, the Sanger Institute reads 60 million base pairs. That’sequal to one of the smaller human chromosomes and many timesthat of an average bacterial genome.

Pathogen Sequencing Unit

http://www.sanger.ac.uk/Projects/Microbes

Bacteria:M. tuberculosisM. lepraeY. pestisS. typhiC. DiphtheriaeBordetella spp. x3B. pseudomalleiS. aureus MRSAS. aureus MSSAE. carrotovora

Yeasts and Fungi:Saccharomyces cerevisiaeSchizosaccharomyces pombeAspergillus fumigatusCandida dubliniensisCandida parapsilosis

Protozoa:Plasmodium falciparum X3Plasmodium spp. X5Leishmania spp.Trypanosoma spp.EimeriaTheileriaBabesia

The Pathogen Group is funded by the Beowulf Genomics Initiativeto sequence the genomes of a wide range of small Eukaryotes and microbes.

Sequencing strategy and assembly

Contiguous sequence

DNA

pUC cloneend sequence

physical gapsequence 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 cloneend sequence

large cloneend sequence

physical gapsequence gap

Shotgun sequencing – strategy

Finished sequence: 100% coverage, 10x depth.

Repeats!!!

Shotgun assembly - Yersinia pestis

PrimaryDNA sequence

Dotter BlastN BlastX

Gene finders

tRNA scan

Repeats Pseudo-genesrRNAGenes

tRNA

Manual curation

PrimaryDNA sequence

Dotter BlastN BlastX

Gene finders

tRNA scan

Repeats Pseudo-genesrRNAGenes

tRNA

Fasta BlastP Pfam Prosite Psort SignalP TMHMM

Manual curation

Manual curation

Annotatedsequence

PSU Projects

Organism

Annotated genome

Finished genome

Database entry

Artemis

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

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 ArtemisUse of multiple lines of evidence

Curating gene models in ArtemisUse of FASTA evidence

EST sequencing & mapping

AAAAAAAAAACAP

AAAAAAAAAACAP

TTTTTTTTT

TTTTTTTTT

intron exon5’UTR Mstop 3’UTR

EST

EST

cDNA

mRNA

ESTs

Curating gene models in ArtemisUse of EST evidence

Curating gene models in ArtemisUse of EST evidence

Curation of gene models in ArtemisMapping proteome fragments to genome

Curation and annotation in ArtemisMapping InterPro domain hits to genome

Finished sequenceFinished sequence

Gene FinderPHAT

GlimmerOrpheus

FASTABLAST

EST

Primary gene modelPrimary gene model

Annotation of pathogen genomes at the PSU (using ARTEMIS)

Complete Annotation Complete Annotation

Organism-specific gene familiesFunctional classification (GO / Riley)

Comparative genomics (using ACT)

Refined gene modelRefined gene model

InterPro scan

HMMPfamHMMSMARTPRINTSPROSITEProDomTIGRFAMs

Manual curationSignalP

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)

Sanger Front page