Stuff to Do

Midterm Iquestions due 1/31

• Email me your question (with answers),– 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,

• 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 ptsmemory only

memory + analysis

integration of information

Cycle SequencingChain Termination

…a DNA polymerase application. ddNTPs

Taq DNA Polymerase w/ Buffer Cycles = Polymerization until Taq hits ddNTP.

dNTPs

Template

Primer

dNTPs

dNTPs and ddNTPS(mixture)

Linked on Course WEB Page.

Cycle Sequence Tutor

…and an animation,http://www.dnalc.org/shockwave/cycseq.html

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

Hierarchical Clone-by-Clone Whole Genome Assembly

Map First: then sequence Sequence First: then map

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.

Clone-by-Clone Ordered Approach

Online Primer: mapping

Genomic Libraries

…how many clones to cover a genome?

Plasmid E. coli up to 15 kb,

Phage E. coli up to 25 kb,

Cosmid E. coli up to 45 kb,

BAC E. coli 100-500 kb,

YAC Yeast 250-1000 kb.

Vectors(carry insert DNA)

plasmid/phage hybrid

Bacterial Artificial

Chromosome

Yeast Artificial Chromosome

Vector Host Inserts

Genomic Sequences and Coverage

N = ln(1 - .9999) ln(1 - v/2,900,000,000)

v = average vector insert size

p finding clone

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

Clone-by-Clone Ordered Approach

Contigs(Contiguos Sequences)

Find overlapping ends…

…Restriction Fragment Length Polymorphisms (RFLPs).

…Sequence,

Clone 1

Clone 2

Sequence Contig

RFLP

Restriction enzymes cut

specific DNA…

…specifically,

Fragment lengths

provide clone

identification data.

Contigs(Contiguos Sequences)

Find overlapping ends…

Find the minimal Tilling Path,

- minimum set of overlapping clones that cover the genome.

Merge good pairs of reads into longer contigs…

Minimal Tilling Path

Fig. 2

Identify minimal overlapping clones.

Shotgun Sequence Each Clone

Bacterial Artificial ChromosomesBACs

• Universal Priming Sites,

– On the vector, flanking the genomic insert.

Shotgun(self-quiz)

~ 8x - 10x coverage:

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?

Contigs

QC

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.

WGA

• Paired End Sequencing,

– sequence both ends of the vector insert, using vector derived primers,

• Maintain mate pair data.

insert

vector

5’

5’3’

3’

Read Pairs = Mate End Pairs

5’ read(543 bp)-atatgtatattgaattacatacatattattaatgcacatttttatccggagttgtggaccatagaaagacatattgactcctcaaagtaaattctgcatgttacattgaaatcataggctaaatttgagatgcactatttttagaaagtgtagagaaaaggacaggaagaaataagcgaaagctttggtaagccaccaaacctgattactggaagaaaagaaaaaagttccgagaatagagttagatcgctggtgagggttttaaatggaacacaacaatggttgttttagagtgtgttattcttttgtatttataccttctcataggtttcttgtaatacacgcttcttcctctctctccctctctcttatggcctcgtcttgaaagcgtcttgcatgctaagagaaggctttagagcaaggagagaagggagaagttgatttatacgtccatcggatatatcttctttttatatctgtctctcttttaaggaagaaaaatggcgactgaattctcgtgggatgaaatcaagaaagaaaatg...

...ggcttgaaatatttggggcaaacaagcttgaagagaaatcagagaacaagtttttgaaattcttggggttcatgtggaatcctctctcatgggttatggagtctgctgcaatcatggctattgttttagctaatggaggaggaaaggcgccggattggcaagattttatcggtattatggtgttgcttatcatcaactccaccataagtttcatcgaggagaacaatgctggcaatgccgctgctgctctcatggcaaatcttgcaccaaagactaaggtatgcaaatttctcaatacatatatataggtatgtattttctaaaaaggagagttatataacctatgtgtgaatgtaggtgttgagagatggtaaatggggggagcaagaggcttcaatcttggttccgggtgatttgataagcatcaaattgggtgacattgttcctgctgatgctcgtctcctcgaaggagatcctttaaaaattgaccaatctgctcttactggtgaatcccttccaaccaccaaacacccaggagat - 3’ read(540 bp)

Example Sequence Output(example: 5 kb insert)

…plus trace data files associated with these sequence runs.

- rest of insert (unsequenced, ~3.9 kb) -

WGA

Structural Genomic Strategies #3 (Hybrid)

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.

WGA

Hybrid

Wednesday

• Please read…Science 291: 1304-1315

• WGA,• Shotgun Sequencing,• Hybrid Approach.

Compartmentalized Shotgun Approach