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Sequence Analysis with Artemis and Artemis Comparison Tool (ACT) Carribean Bioinformatics Workshop 18 th -29 th January , 2010. atcttttacttttttcatcatctatacaaaaaatcatagaatattcatcatgttgtttaaaataatgtattccattatgaactttattacaaccctcgtt - PowerPoint PPT Presentation
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Sequence Analysis with Artemisand
Artemis Comparison Tool (ACT)
Carribean Bioinformatics Workshop
18th-29th January , 2010
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Sequencing is just the beginning of the process
Extracting information & interpreting
What´s therewhere are the geneswhich geneshow to find them?
SEQUENCE ANNOTATION
Sequencing is just the beginning of the process
Extracting information & interpreting
What´s therewhere are the geneswhich geneshow to find them?
SEQUENCE ANNOTATION
Strategies for sequence annotationStrategies for sequence annotation
Predictive methods
Comparative methods
Experimental methods
Interpretation of the DNA sequence into genes according to rules
Strategies for sequence annotationStrategies for sequence annotation
Predictive methods
Comparative methods
Experimental methods
Interpretation of the DNA sequence into genes according to rules
Interpretation of the DNA sequence into genes according to similarities with other sequences
Strategies for sequence annotationStrategies for sequence annotation
Predictive methods
Comparative methods
Experimental methods
Interpretation of the DNA sequence into genes according to rules
Interpretation of the DNA sequence into genes according to similarities with other sequences
Interpretation of the DNA sequence into genes according to experimental results (e.g. cDNA)
EST Blast Hit
Gene prediction programs:ORFs and CDSs
ORFs are not equivalent to CDSs
Not all open reading frames are coding sequences
Gene prediction
Gene finder
Glimmer
Orpheus PHAT
GeneMark
Gene finding programs
• Genefinding software packages use Hidden Markov Models.
• Predict coding, intergenic and intron sequences
• Need to be trained on a specific organism.
• Never perfect!
Gene prediction programs: Problems
• ORFs are not equivalent to CDSs
• Gene prediction programs find new genes that share properties with a given set of genes.
• They can be confounded by:– Sequence constraints (ribosomal proteins etc.)
– Sequence biases
– Different sets of genes
– Horizontal gene transfer
– Non-coding DNA
Gene prediction programs: Problems
Different gene training sets: Plasmodium falciparum
Original annotation
Updated annotation
Gene prediction programs: Problems
Non-protein coding regions: S. typhi ribosomal RNA genes
glimmer
genefinder
final
orpheus
glimmer
genefinder
final
orpheus
Gene prediction programs: ProblemsNon-protein coding regions: N. meningitidis DNA repeats
glimmerorpheusfinal
glimmerorpheusfinal
Gene prediction programs: Problems
Pseudogenes M. leprae
Gene prediction programs: Problems
Pseudogenes: M. lepraeGlimmer
Gene prediction programs: Problems
Pseudogenes: M. lepraePseudogenes: M. lepraeORPHEUS
Gene prediction programs: Problems
Pseudogenes: M. leprae
WUBLASTX vs. M. tuberculosis
Gene prediction programs: Problems
Pseudogenes: M. leprae
Final annotation
The Gene Prediction Process
DNA SEQUENCE
AN
NA
LY
SIS
SO
FT
WA
RE
UsefullCDSPrediction
Annotator
AT content
Gene finders
Codon Usage
BlastX
FASTA
ESTs
Eukaryotic gene
AAAAAAAAAACAP
AAAAAAAAAACAP
TTTTTTTTT
TTTTTTTTT
intron Exon II5’UTR Exon Istop
3’UTR
EST
cDNA
mRNA
EST
Exon III
ATG GT AG GT AG
AT content• Coding regions have higher GC content in
AT rich genomes
AT content
CODON USAGE
• Codon bias is different for each organism.• DNA content in coding regions is restricted
– but it is not restricted in non coding regions.
• The codon usage for any particular gene can influence expression.
Codon usage
• All organisms have a preferred set of codons.
Malaria TrypanosomaGUU 0.41 GUU 0.28
GUC 0.06 GUC 0.19
GUA 0.42 GUA 0.14
GUG 0.11 GUG 0.39
Codon Usage• http://www.kazusa.or.jp/codon/
Codon Usage in Artemis
Forward frames
Reverseframes
Codon usage & gene finding in : Leishmania
GC frame plot
• Plots the third position GC content of each frame of a DNA sequence.
• In coding DNA the GC content of the 3rd base is often higher.
• Good prediction of coding in malaria and trypanosomes.
GC frame plot of tubulin gene cluster on T. brucei Chr 1
Homology Data
• Coding regions are more conserved than non coding regions due to selective pressure.
• Comparing all possible translations against all known proteins will give clues to known genes.
• Blastx
Gene finding: using ACT
TBLASTX comparisons
P. knowlesi
P. falciparum
P. yoelii
Gene finding by RNA-Seq(Transcriptional landscape of Neospora caninum Tachyzoites
Day 3 Tachyzoites (RNAseq)
Day 4 Tachyzoites (RNAseq)
Day 3 Tachyzoites (RNAseq)
Day 4 Tachyzoites (RNAseq)
N. caninum Chr08
T. gondii Chr085’ UTR 3’ UTR
TBLASTX matches visualised in ACT
Transcriptome sequencing in Neospora(RNAseq is useful for predicting/confirming UTR boundaries)
RNA-Seq: correcting gene models
Before
%GC
After
%GC
__16hr, __32hr, __48hr
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