1. Human Genome

2. Human Genome Contents: 3200 Mb
Genes: 1200 Mb
Genes 48 Mb
Related 1152 Mb: Pseudogenes, Gene Fragments, Introns
Intergenic DNA 2000 Mb
Interspersed Repeats 1400 Mb
Microsatellite (short tandem repeats) 90 Mb
Telomeres: End Sequences
Centromeres:
Single Nucleotide Polymorphisms

3. Chromosomes Shorter than DNA they contain
Histones: DNA binding proteins
Two Copies held together by centromeres
Telomere: Terminal region
Two humans differ by 0.1%

5. Donors HGP: Celera: 5 subjects (three men; two women)
Opportunity advertised near labs
First come; First Taken
5-10 samples for every one used
No link between donor and sample
Celera: 5 subjects (three men; two women)
One Asian; One African-American; One Hispanic; Two Caucasians
Craig Venter

6. Basic Technology Physical Mapping Cloning Shotgun Sequencing
Computational Sequence Reassembly

7. STS High Resolution, Rapid, Simple 100 - 500 bp
Collection of overlapping fragments
Each point represented multiple times in random fragments
Sequence must be known
Unique in chromosome under study

8. Physical Mapping
A set of clone fragments whose position relative to each other is known
Restriction Maps: Relative locations of Restriction Sites
Fluorescent in situ hybridization (FISH): Marker locations mapped by hybridizing probe to chromosomes
Sequence Tagged Sites (STS): Positions of short sequences mapped by PCR or hybridization analysis of genome fragments
Expressed Sequence Tags (EST): short sequences from cDNA clones

9. Genome cut into fragments
Cloned as library in vector (red)

10. Hybridisation mapping:1 pick clones into a grid
2 hybridise to probe 1
3 hybridise to probe 2
4 build contigs In this case, two clones
hybridised to both probes and thus they are
predicted to overlap. Those hybridising to only one probe are predicted to extend out to the left or right.

11. Fingerprinting:
Digest clones and run
On gel
Overlap by shared bands

12. Assembly of Contiguous DNA Sequence
Shotgun Approach
Contigs: Result of joining overlapping sequences
Scaffold: Result of connecting contigs by filling in gaps
BAC: Bacteria artificial chromosome vector: Inserts kbs

13. Regional mapping

14. Regional mapping

15. Regional mapping
Minimal tiling path selected for sequencing.

16. Restriction fragment fingerprinting Molecular weight marker every
5th lane
Restriction
fragment
fingerprinting
>20 kbp
~300 bp
- BAC clones are grown
in 96-well format
- Hind III digest
- 1% agarose

17. Contig assembly FPC* Overlap identification by
restriction pattern similarities
Facilitated contig assembly
*Sanger Centre
C. Soderlund, I Longden and R. Mott
Clone A B C D E F G *
All restriction fragments within
a clone selected for the tiling
path must be verified by their
presence in overlapping clones.
: insert fragments
: vector fragments

18. BCM-
HGSC

19. Shotgun Sequencing I :RANDOM PHASE
Sheared DNA: kb
Bac Clone: kb
Random
Reads
Sequencing
Templates:
BCM-
HGSC

20. Shotgun Sequencing II:ASSEMBLY
Low Base
Quality
Single
Stranded
Region
Mis-Assembly
(Inverted)
Sequence
Gap
Consensus
BCM-
HGSC

21. Shotgun Sequencing III: FINISHING
Low Base
Quality
Single
Stranded
Region
Mis-Assembly
(Inverted)
Sequence
Gap
Consensus
BCM-
HGSC

22. Shotgun Sequencing III: FINISHING
Single
Stranded
Region
Mis-Assembly
(Inverted)
Sequence
Gap
Consensus
BCM-
HGSC

23. Shotgun Sequencing III: FINISHING
Mis-Assembly
(Inverted)
Sequence
Gap
Consensus
BCM-
HGSC

24. Shotgun Sequencing III: FINISHING
Mis-Assembly
(Inverted)
Consensus
BCM-
HGSC

25. Shotgun Sequencing III: FINISHING
High Accuracy Sequence:
< 1 error/ 10,000 bases
BCM-
HGSC

26. Whole Genome Shotgun Sequencing
Sheared DNA: kb
Whole Genome:
3,000 Mb
Random
Reads
Sequencing
Templates:
BCM-
HGSC

27. Whole Genome Shotgun Sequencing:Assembly
Low Base
Quality
Single
Stranded
Region
Mis-Assembly
(Inverted)
Sequence
Gap
Consensus
BCM-
HGSC

28. Whole Genome Shotgun Sequencing:Assembly
Sequence
Gap
Low Base
Quality
Consensus
BCM-
HGSC

29. Random fragmentation of genome produces good sampling of its sequence space. Overlaps are identified, and subassembly of sequence takes place after cloning into universal vector.

30. Digested into Random Fragments

31. Cloned into Vector

32. Sequenced from know ends of plasmid (vector)

33. Assembled into contigs
Assembled into contigs. Gaps and single-stranded regions identified for further study. Targeted for new sequencing. Double-Barreled: Both Strands.

34. In the gaps:

36. Whole-Genome Shotgun Sequencing
Speed-up: Assembled Correctly?
Avoid up-front mapping
Huge amount of computer time to identify overlaps
Have to reference a map
Repeats are a problem:
Leave out sequence between repeats
Missing Reference End Sequence means Error

38. HGP
Isolate large fragments in BACs with framework of landmark-based physical map
Sequence on clone-by-clone basis
Time-Consuming subcloning of random fragments and physical mapping

40. Sequence Reassembly Phrap Shortest Covering Superstring Map Assembly
Overlap: Finding overlapping fragments
Layout: ordering fragments
Consensus: Sequences from layout