This presentation elaborates the genome annotation. This presentation presented at PMAS-UAAR, Pakistan. Delivered by: MUHAMMAD TAJAMMAL KHAN

1. Genome Annotation
Delivered by
Muhammad Tajammal Khan
M.Phil (Botany)
11-arid-3759

2. Definition: Genome Annotation is the process of
interpreting raw sequence data into useful biological
information Annotations describe the genome and
transform raw genome sequences into biological
information by integrating computational analyses,
other biological data and biological expertise.

3. Unannotated DNA
5' 3'
Annotated DNA
Legend:
Exon (protein coding)
Intron
Intergenic sequence

4. Annotation may be
Structural annotation
ORFs and their localisation (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)
gene structure
coding regions
location of regulatory motifs
Functional annotation
biochemical function
biological function
involved regulation and interactions
expression

5. Things we are looking to
annotate?
 CDS
 mRNA
 Promoter and Poly-A Signal
 Pseudogenes
 ncRNA

6. Tools
 ORF detectors
◦ NCBI: http://www.ncbi.nih.gov/gorf/gorf.html
 Promoter predictors
◦ CSHL: http://rulai.cshl.org/software/index1.htm
◦ BDGP: fruitfly.org/seq_tools/promoter.html
◦ ICG: TATA-Box predictor
 PolyA signal predictors
◦ CSHL: argon.cshl.org/tabaska/polyadq_form.html
 Splice site predictors
◦ BDGP:
http://www.fruitfly.org/seq_tools/splice.html
 Start-/stop-codon identifiers
◦ DNALC: Translator/ORF-Finder
◦ BCM: Searchlauncher

7. Overview of
genome
analysis

8. Two approaches to genome sequencing
Whole Genome Shotgun
An approach used to decode an organism's genome
by shredding it into smaller fragments of DNA which
can be sequenced individually. The sequences of these
fragments are then ordered, based on overlaps in the
genetic code, and finally reassembled into the complete
sequence. The 'whole genome shotgun' (WGS) method is
applied to the entire genome all at once, while the
'hierarchical shotgun' method is applied to large,
overlapping DNA fragments of known location in
the genome.

9. Two approaches to genome sequencing
Hierarchical shotgun method
Assemble contigs from various chromosomes, then sequence and assemble
them. A contig is a set of overlapping clones or sequences from which a
sequence can be obtained.
A contig is thus a chromosome map showing the locations of those regions of
a chromosome where contiguous DNA segments overlap. Contig maps are
important because they provide the ability to study a complete, and often
large segment of the genome by examining a series of overlapping clones
which then provide an unbroken succession of information about
that region.

10. Hierarchical vs. Whole Genome

11. Sequencing technology in 12 steps

12. 1. Prepare genomic DNA
2. Attach DNA to surface
DNA 3. Bridge amplification
4. Fragments become
adapters
double stranded
5. Denature the double-
stranded molecules
6. Complete amplification
Randomly fragment genomic DNA and ligate
adapters to both ends of the fragments

13. adapter
DNA 1. Prepare genomic DNA
fragment
2. Attach DNA to surface
dense lawn 3. Bridge amplification
of primers
adapter 4. Fragments become
double stranded
5. Denature the double-
stranded molecules
6. Complete amplification
Bind single-stranded fragments randomly to
the inside surface of the flow cell channels

14. 1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become
double stranded
5. Denature the double-
stranded molecules
6. Complete amplification
Add unlabeled nucleotides and enzyme to
initiate solid-phase bridge amplification

15. 1. Prepare genomic DNA
2. Attach DNA to surface
Attached 3. Bridge amplification
Attached terminus free terminus
terminus 4. Fragments become
double stranded
5. Denature the double-
stranded molecules
6. Complete amplification
The enzyme incorporates nucleotides to
build double-stranded bridges on the
solid-phase substrate

16. 1. Prepare genomic DNA
2. Attach DNA to surface
Attached 3. Bridge amplification
Attached
4. Fragments become
double stranded
5. Denature the double-
stranded molecules
6. Complete amplification
Denaturation leaves single-
stranded templates anchored to
the substrate

17. 1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become
double stranded
5. Denature the double-
stranded molecules
Clusters
6. Complete amplification
Several million dense clusters of
double-stranded DNA are generated in
each channel of the flow cell

18. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second
chemistry cycle
11. Sequencing over
multiple chemistry cycles
12. Align data
Laser
The first sequencing cycle begins by
adding four labeled reversible terminators,
primers, and DNA polymerase

19. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second
chemistry cycle
11. Sequencing over
multiple chemistry cycles
12. Align data
After laser excitation, the emitted
fluorescence from each cluster is captured
and the first base is identified

20. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second
chemistry cycle
11. Sequencing over
multiple chemistry cycles
Laser 12. Align data
The next cycle repeats the incorporation
of four labeled reversible terminators,
primers, and DNA polymerase

21. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second
chemistry cycle
11. Sequencing over
multiple chemistry cycles
12. Align data
After laser excitation the image is
captured as before, and the identity of
the second base is recorded.

22. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second
chemistry cycle
11. Sequencing over
multiple chemistry cycles
12. Align data
The sequencing cycles are repeated to
determine the sequence of bases in a
fragment, one base at a time.

23. Reference
sequence
7. Determine first base
8. Image first base
9. Determine second base
10. Image second
chemistry cycle
11. Sequencing over
multiple chemistry cycles
12. Align data
The data are aligned and compared to
a reference, and sequencing
differences are identified.

24. The generic structure of an automatic genome annotation pipeline and
delivery system

25. The Annotation Process
ANNALYSIS SOFTWARE
DNA SEQUENCE
Useful
Information
Annotator

26. Annotation Process
DNA sequence
RepeatMasker Blastn Gene finders Blastx Halfwise tRNA scan
Repeats Promoters rRNA Pseudo-Genes tRNA
Genes
Fasta BlastP Pfam Prosite Psort SignalP TMHMM

27. Genome Browsers
Generic Genome Browser (CSHL) NCBI Map Viewer Ensembl Genome Browser
www.wormbase.org/db/seq/gbrowse www.ncbi.nlm.nih.gov/mapview/ www.ensembl.org/
UCSC Genome Browser Apollo Genome Browser
genome.ucsc.edu/cgi-bin/hgGateway?org=human www.bdgp.org/annot/apollo/

28. What is gene
prediction?
Detecting meaningful signals in uncharacterised DNA sequences.
Knowledge of the interesting information in DNA.
GATCGGTCGAGCGTAAGCTAGCTAG
ATCGATGATCGATCGGCCATATATC
ACTAGAGCTAGAATCGATAATCGAT
CGATATAGCTATAGCTATAGCCTAT
 Gene prediction is ‘recognising protein-
coding regions in genomic sequence’

29. Basic Gene Prediction Flow
Chart
Obtain new genomic DNA sequence
1. Translate in all six reading frames and compare to protein
sequence databases
2. Perform database similarity search of expressed sequence tag
Sites (EST) database of same organism, or cDNA sequences if
available
Use gene prediction program to locate genes
Analyze regulatory sequences in the gene

30. Approaches to gene prediction
Ab Initio Gene Finding
http://exon.gatech.edu/GenMark/eukhmm.cgi
http://sun1.softberry.com/berry.phtml=fgenesh&group=programs
&subgroup=gfind
Repeat Masking
http://www.repeatmasker.org
http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker
Transcript based prediction
http://plantta.tigr.org
http://harvest.ucr.edu/
Gene function CDNA
http://au.expasy.org/sprot/
http://www.pir.uniprot.org/
Gene Ontologies
http://www.geneontology.org

31. Visualization Tools
http://www.gmod.org/?q=node/4
http://www.gmod.org/?q=node/71

32. Prediciton of Secondary Structure and Folding Classes
• nnpredict http://www.cmpharm.ucsf.edu/_nomi/nnpredict.html
• PredictProtein http://www.embl-heidelberg.de/predictprotein/
• SOPMA http://pbil.ibcp.fr/
• Jpred http://jura.ebi.ac.uk:8888/
• PSIPRED http://insulin.brunel.ac.uk/psipred
• PREDATOR http://www.embl-heidelberg.de/predator.html
Prediction of Specialized Structures or Features
• COILS http://www.ch.embnet.org/software/COILSform.html
• MacStripe www.york.ac.uk/depts/biol/units/coils/mstr2.html
• PHDtopology http://www.embl-heidelberg.de/predictprotein
• SignalP http://www.cbs.dtu.dk/services/SignalP/
• TMpred http://www.isrec.isb-sib.ch/ftp-erver/tmpred
www/TMPREDform.html
Structure Prediction
• DALI http://www2.ebi.ac.uk/dali/
• Bryant-Lawrence ftp://ncbi.nlm.nih.gov/pub/pkb/
• FSSP http://www2.ebi.ac.uk/dali/fssp/
• UCLA-DOE http://fold.doe-mbi.ucla.edu/Home
• SWISS-MODEL http://www.expasy.ch/swissmod/SWISS-MODEL

33. Search using the gene
name

34. Click the name for transcript
info

35. Click the contig to see detailed information

36. Click the contig to see detailed information
Click here to export the contig

38. Select output format, then click Continue

39. Save or copy the data for further analysis

40. Genomic region

41. Coding sequence

42. Click here for pairwise BLAST

43. Click ‘Align’ to
proceed
Paste in genomic sequence
Paste in CDS sequence

47. This is a protein BLAST
(BLASTP)

49. Some Concluding remarks
 Trust but verify
 Beware of gene prediction tools!
 Always use more than one gene
prediction tool and more than one
genome when possible.
 Active area of bioinformatics research,
so be mindful of the new literature in
this .