What data

1
What data?

• Today Structural Genomics
• Friday Data Base Resources

2
Structural Genomics

• Characterizing and locating the entire set of
  genes in a genome.

3
Structural Genomic Strategies 1

• Ordered 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.
• heavy up-front demand for human labor.

4
Sequence Ready Ordered Approach
Stitch, then sequence.
5
Structural Genomic Strategies 2

• Shotgun Approach
• Sequence first, then order,
• dependent on advances in computer analysis and
  sequencing technologies,
• dependent on automated labor.

6
Genomes...T.A. Brown

• The big question is whether the 70 million
  sequences could be assembled correctly...
• ...if the conventional shotgun approach is used,
  which makes no reference to a genome map, then
  the answer is certainly no.

Wrong, the answer is Yes, but the truth is,
with a map the sequence is better.
7
First the Genome(s) Celera

• 5 individuals
• two males / three females
• one African American
• one Asian Chinese
• one Hispanic Mexican
• two Caucasians
• take tissue and sperm samples, immortilize,
• extract DNA,
• shred and package in vectors.

8
Bacterial Artificial ChromosomesBACs

• F plasmid ancestry,
• maintain bacterial replication system and copy
  number control system.

9
Science 291 (5507), 1304-1351
8, September 1999 - 25, June 2000
10
Single Strand PCR
dNTPs
5 - ATACATACTACTAACTAACTAA - 3
3 - TATGTATGATGATTGATTGATT
- 5
Template
1 Primer
Taq Polymerase w/ Buffer
Cycles

Polymerization until Taq falls off, linear
amplification.
11
Cycle SequencingChain Termination
ddNTPs
dNTPs
5 - ATACATAC - 3
3 - TATGTATGATGATTGATTGATT - 5
Template
1 Primer
Taq Polymerase w/ Buffer
Cycles

Polymerization until Taq hits ddNTP, linear
amplification.
12
Fluorescent ddNTPs
13
(No Transcript)
14
ABI 3700

• Automated,
• Capillary Action,
• 15 minutes a day maintenance,
• 65 full-time staff.

15
Systems Biology
16
Mate Pairs

• BAC End Sequencing,
• sequence both ends of the BAC using vector
  derived primers.

17
Science 291 (5507), 1304-1351
8, September 1999 - 25, June 2000
18
(No Transcript)
19
Whole Genome Assembly
20
WGA

• 1. Screener
• 2. Overlapper
• 3. Unitigger,
• 4. Scaffolder,
• 5. Repeat Resolver.

21
Screener

• ...finds and masks microsatellite repeats,
  known repeat regions and ribosomal DNA,
• marks the rest for overlapping.

22
Overlapper

• ...looks for end-to end overlaps of at least 40
  bp with no more than 6 differences in match.

Whats the significance?
...a one in 1017 event.
23
But(t)!

• ...the Screener doesnt include all of the low
  frequency level repeats,
• ...so, a majority of the Overlapper outputs are
  bogus.

24
What Now?

• ...some uniquely assembled contigs (unitigs) are
  readily identifiable,
• all of the assembled sequences match over all of
  the known sequence,

- and -

• ...are consistent with an 8x coverage,
• over-collapsed assemblies are identified and
  broken down into unitigs when possible.

25
Scaffolder

• ...contigs the contigs,
• uses mate-pair information.

26
Repeat Resolver

• ...most of the remaining gaps were due to repeats.

...91 sequence, 9 gaps,

• Gaps,
• average 2.43 kb,
• over 50 lt 500 bp,
• over 62 lt 1 kb,
• no gap larger than 100 kb.

27
Scaffolds
28
Friday

• Databases,
• Database Mining.