Genome Characterization

1
Genome Characterization

• DNA sequence-ULTIMATE Map
• DNA sequencing-methods
• Assembly/sequencing

Assigned reading Ch 13, Service 2006 review paper
BIO520 Bioinformatics Jim Lund
2
DNA Sequence Project Size/Type

• 500 bases
• 2500 bases
• 10 kbp
• 150 kbp
• 3 Mbp
• simple
• repeats
• 3 Gbp
• 31 Gbp

• 1 locus EST,STS
• whole cDNA/EST
• Gene, virus
• BAC, big virus
• Bacterial genome, YAC-size
• Human, mouse
• Salamander

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Genome sizes

• Nematode (Caenorhabditis elegans) 100 Mb
• Thale cress (Arabidopsis thaliana) 160 Mb
• Fruit fly (Drosophila melanogaster) 180 Mb
• Puffer fish (Takifugu rubripes) 400 Mb
• Rice (Oryza sativa) 490 Mb
• Human (Homo sapiens) 3.5 Gb
• Leopard frog (Rana pipiens) 6.5 Gb
• Onion (Allium cepa) 16.4 Gb
• Mountain grasshopper(Podisma pedestris) 16.5
  Gb
• Tiger salamander (Ambystoma tigrinum) 31 Gb
• Easter lily (Lilium longiflorum) 34 Gb
• Marbled lungfish (Protopterus aethiopicus) 130 Gb

4
DNA Sequencing Methods

• Chain termination/Dideoxy/Sanger
• Fluorescence paradigm, ABI
• Main method
• Sequencing by hybridization (SBH)
• Chips Affymetrix (Lander, et al)
• Other formats
• Hyseq

5
Sequence by Hybridization

• High throughput, highly parallel Many different
  formats
• Superb for limited regions of genome

GAGCTACGTGACACGTCAGTCCCAG GAGCTACGTGACCCGTCAGTCCCA
G GAGCTACGTGACGCGTCAGTCCCAG GAGCTACGTGACTCGTCAGTCC
CAG
A
C
T
G
T
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Dideoxy/Chain Terminator/Sanger

• Template
• Primer
• Extension Chemistry
• polymerase
• termination
• labeling
• Separation
• Detection

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Chain Terminator Basics
TGCA
Extend
dN ddN 100 1
Ladder n, n1...
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Electrophoresis
Sequencing Reaction products
Polyacrylamide Gel Electrophoresis (PAGE)
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Separation

• Gel Electrophoresis
• Capillary Electrophoresis
• suited to automation
• rapid (2 hrs vs 12 hrs)
• re-usable
• simple temperature control
• 96 well format

migration 1/log N
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Paradigm Instrument

• Applied Biosystems
• http//www.appliedbiosystems.com/
• ABI3730XL (2002, 96 samples, 1000 base reads,
  350,000, higher sensitivity, lower reagent
  cost, 1/reaction)
• 700 Kbp / 24 hours.
• 384 capillary sequencers
• 5700 sequences / 24 hr day
• 2.8 Mbp / 24 hours.

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384-well capillary sequencing
Results are shown as an electropherogram showing
a peak for each base. From the peak heights and
widths, a Phred score is assigned to each
individual base. A high Phred score indicates a
high certainty as to the identity of that
particular base.
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Sample Output
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Limitations/Challenges

• 1 trace1000 bases or less
• How do we cover a genome?
• DIVIDE AND CONQUER assemble these short sequence
  fragments.

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Trace Editing

• EditView (ABI PRISM)
• Mac
• Chromas (free/pay versions)
• Windows
• Consed
• UNIX
• VectorNTI Contig Express

15
Sequencing Strategies

• Ordered
• Divide and Conquer
• Random Sequence
• Brute Force

Sequencing Assembly Finishing Annotation
Big projects often mix methods.
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Random Method

• Shear DNA (nebulize)
• finish ends, ligate into vector
• Produce template
• Sequence to 8X 10X coverage
• Sequence both ends of templates.
• Read length (500 typical)
• Accuracy (99 good)

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Assembly Problem
CONTIG
18
Contigs, Islands
contigs
Island
19
Random
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Continuing rapid improvement in sequencing
technology
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• 1990s Human genome 3Gbps, 300 million (just
  sequencing)
• Current Mammalian genome (3 Gbps) 30 million
• Goal 100,000 genome, 300X cheaper (and
  faster)

Current best technology