1. Stuff to Do

2. Midterm I questions due 1/28
me your question (with answer),
if you have the capability, mail complete questions, figures, etc. and all,
if not, write questions, with instructions…i.e. in Figure 2 of x paper, blah, blah, blah,
That Friday afternoon, I’ll post the questions on the WEB page, on Monday, you’ll have time to work on them together, in class.
0 pts
5 pts
memory only
memory +
analysis
integration of information

3. Cycle Sequencing Chain Termination …a DNA polymerase application.
ddNTPs
dNTPs
Template
Primer
Taq DNA Polymerase w/ Buffer
Cycles =
Polymerization until Taq hits ddNTP.

4. dNTPs
dNTPs and ddNTPS
(mixture)

5. Linked on Course WEB Page.
Cycle Sequence Tutor
…and an animation,

6. Disclaimer: this review is heavily biased toward the public sequencing consortium.

7. Hierarchical Clone-by-Clone
Whole Genome Assembly
Map First: then sequence
Sequence First: then map

8. Genome Sequencing Strategy #1
Clone-by Clone Approach
Order clones along the genome, then sequence,
not dependent on acceleration of sequencing capacity,
not dependent on advanced computer analysis,
not dependent on ‘as-of-yet’ sequencing technologies,
“repeats” not as big a problem?
heavy up-front demand for human labor.

9. Clone-by-Clone Ordered Approach
Online Primer: mapping

10. Genomic Libraries
…how many clones to cover a genome?

11. Vectors (carry insert DNA)
Host
Inserts
Plasmid E. coli up to 15 kb,
Phage E. coli up to 25 kb,
Cosmid E. coli up to 45 kb,
BAC E. coli kb,
YAC Yeast kb.
plasmid/phage hybrid
Bacterial Artificial Chromosome
Yeast Artificial Chromosome

12. Genomic Sequences and Coverage
p finding clone
N = ln( )
ln(1 - v/2,900,000,000)
v = average vector insert size
genome
size
plasmid (5 kb) = 5.3 x 106
phage (20 kb) = 1.3 x 106
BAC (125 kb) = 2.2 x 105
YAC (500 kb) = 27,000 clones

13. Clone-by-Clone Ordered Approach

14. Contigs (Contiguos Sequences)
Find overlapping ends…
Clone 1
Clone 2
…Sequence,
…Restriction Fragment Length Polymorphisms (RFLPs).

15. Sequence Contig

16. Restriction enzymes cut
RFLP
Restriction enzymes cut
specific DNA…
…specifically,
Fragment lengths
provide clone
identification data.

18. Contigs (Contiguos Sequences)
Find overlapping ends…
Merge good pairs of reads into longer contigs…
Find the minimal Tilling Path,
- minimum set of overlapping clones that cover the genome.

19. Identify minimal overlapping clones.
Minimal Tilling Path
Fig. 2
Identify minimal overlapping clones.
Shotgun Sequence Each Clone

20. Bacterial Artificial Chromosomes BACs
Universal Priming Sites,
On the vector, flanking the genomic insert.

21. Shotgun (self-quiz) ~ 8x - 10x coverage: Why Shotgun?
To shotgun sequence 10,000 bp, you’d need 80k - 100k bp of sequence, or ~160 - ~180 sequencing reactions.
But, 10,000 bp, at 500 bp per sequencing reaction could be done in as few as 20 sequencing reactions.
Why Shotgun?

22. Contigs QC

24. Structural Genomic Strategies #2
Whole Genome Assembly Approach:
Sequence first, then order,
dependent on advances in computer analysis and sequencing technologies,
dependent on automated labor.

25. WGA

26. Read Pairs = Mate End Pairs
Paired End Sequencing,
sequence both ends of the vector insert, using vector derived primers,
Maintain mate pair data.
insert
vector 5’ 3’ 3’ 5’

27. Example Sequence Output (example: 5 kb insert)
5’ read(543 bp)-atatgtatattgaattacatacatattattaatgcacatttttatccggagttgtggaccatagaaagacatattgactcctcaaagtaaattctgcatgttacattgaaatcataggctaaatttgagatgcactatttttagaaagtgtagagaaaaggacaggaagaaataagcgaaagctttggtaagccaccaaacctgattactggaagaaaagaaaaaagttccgagaatagagttagatcgctggtgagggttttaaatggaacacaacaatggttgttttagagtgtgttattcttttgtatttataccttctcataggtttcttgtaatacacgcttcttcctctctctccctctctcttatggcctcgtcttgaaagcgtcttgcatgctaagagaaggctttagagcaaggagagaagggagaagttgatttatacgtccatcggatatatcttctttttatatctgtctctcttttaaggaagaaaaatggcgactgaattctcgtgggatgaaatcaagaaagaaaatg...
- rest of insert (unsequenced, ~3.9 kb) -
...ggcttgaaatatttggggcaaacaagcttgaagagaaatcagagaacaagtttttgaaattcttggggttcatgtggaatcctctctcatgggttatggagtctgctgcaatcatggctattgttttagctaatggaggaggaaaggcgccggattggcaagattttatcggtattatggtgttgcttatcatcaactccaccataagtttcatcgaggagaacaatgctggcaatgccgctgctgctctcatggcaaatcttgcaccaaagactaaggtatgcaaatttctcaatacatatatataggtatgtattttctaaaaaggagagttatataacctatgtgtgaatgtaggtgttgagagatggtaaatggggggagcaagaggcttcaatcttggttccgggtgatttgataagcatcaaattgggtgacattgttcctgctgatgctcgtctcctcgaaggagatcctttaaaaattgaccaatctgctcttactggtgaatcccttccaaccaccaaacacccaggagat - 3’ read(540 bp)
…plus trace data files associated with these sequence runs.

28. WGA

29. Structural Genomic Strategies #3 (Hybrid)

30. Project Comparisons (NYT: 10/3/2002)
Decoding the genome of Plasmodium falciparum, the most dangerous of the four single-cell parasites that cause malaria, took six years and cost about $20 million, paid for by the Wellcome Trust of London, the National Institutes of Health in Bethesda, Md., and other sources. Dr. Malcolm J. Gardner of the Institute for Genomic Research in Rockville, Md., led a large team of scientists there and at the Sanger Centre near Cambridge in England. Completion of the falciparum genome was first announced at a conference in Las Vegas in February.
The genome of Anopheles gambiae, the primary carrier of the parasite, was begun more recently and took a mere 15 months even though its genome is far larger — some 278 million units of DNA encoding 14,000 genes compared with the parasite's 23 million units of DNA and 5,268 genes. The mosquito team was led by Dr. Robert A. Holt of Celera Genomics in Rockville. The $14 million cost was born by the National Institutes of Health, by Genoscope in France and other sources.
Hybrid
WGA

31. Next WGA, Shotgun Sequencing, Hybrid Approach. Compartmentalized
Please read…
Science 291: