Genome Characterization

1
Genome Characterization

• Sequencing, contd
• Physical Mapping

2
Assembly Problem
CONTIG
3
Contigs, Islands
contigs
Island
4
Assembly Methods

• Strip out vector
• Mask known repeats
• Trim off unreliable data
• Find Matches (500 x 500 x many!!)
• how long (and what ktuple)
• how perfect (reliability index)
• where to look? (ends only vs entire)

5
Assembly Programs

• PHRAP FAMILY
• phrap, kangaroo, phrapo,
• GAP4, TIGRAssembler,...
• GCG
• gelstart, gelenter, gelmerge, gelassemble,
  geldisassemble
• thinly veiled vi editor
• SeqWeb.

6
Project Management

• Editing and Assembly
• RepeatMasker
• Phred/Phrap
• Consed
• Databases
• ACeDB
• A C. elegans database
• Oracle

7
Annotation

• ORFs
• GRAIL, PowerBLAST
• Other data?
• Repeats
• Other Regions

Submit to Genbank ...HTGS (level1,2,3) ...nr
8
Sequencing by Hybridization
Hybridize labeled query DNA
CHIP OLIGOS (20-mers)
...gaactAatact... ...gaactCatact... ...gaactGatact
... ...gaactTatact...
site 1
...gaactaAtact... ...gaactaCtact... ...gaactaGtact
... ...gaactaTtact...
site 2
GAACTATGTACT
9
Modern Sequencing Challenges

• Heterozygous DNAs
• germline differences
• somatic variation
• Massive sequencing
• population studies
• genome scans
• Minimal sample preparation
• Doctors Office

Chips, Quantitative Seq Automation Miniaturization
10
Physical Mapping

• Divide and Conquer
• Physical vs Biological Mapping

11
Physical Mapping
12
Clone-based Maps

• Genome fragmentation and cloning
• DNA separation
• vectors, etc.
• Physical map assembly
• hybridization
• fingerprinting

13
Genome Fragmentation

• Restriction digestion
• partial digestion
• modified digestion
• methylase, restriction
• Physical fragmentation
• Others?

14
Separation of Large DNAs

• Pulsed Field Gel Electrophoresis
• Chromosome Sorting
• FACS
• Microdissection
• Somatic Cell Hybrids

15
PFGE

• Formats
• CHEF, TAFE
• Limitations
• 10 Mbp

16
Cloning Vectors

• YAC(Yeast Artificial Chromosomes)
• BAC (Bacterial Artificial Chromosomes)
• PAC
• Cosmids
• ? phage
• Plasmids
• M13
• physical clones

17
YAC
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YACs, contd

• 1-3 Mbp possible
• Problems
• specific instabilities
• low DNA yield
• difficult to manipulate

19
BACs, PACs

• BACmini-F plasmid
• low copy, good stability
• 150 kb size
• low yield (hard to sequence, but possible)
• size easy to manipulate
• size sufficient for human DNA repeat structure
• PACP1 replicon

20
Cosmids,?, plasmids

• Cosmids 40 kbp
• subject to rearrangement, instability
• easy to manipulate
• ? 20 kbp
• plasmids

21
M13, phagemids, physical clones

• easy to manipulate
• easy to sequence
• least useful biologically

22
Overlaps/Assembly

• Hybridization
• Fingerprinting

23
Hybridization

• Ordered Arrays of Clones
• Hybridize with chromosomes
• bins cosmids
• identifies repeats
• Select single cosmid, use as probe
• Generate overlaps by sampling

Draw
24
Fingerprinting

• Restriction nuclease digestions
• BamHI, EcoRI, etc.
• Shared vs Unshared fragments

Draw
25
Assembly ImprovementsRepeat Problems

• Multiple fragment sizes in 1 project
• Use length/distance info