DNA Sequencing

1
DNA Sequencing

• Basic Techniques
• Project Design
• Process Improvements

2
Project Size/Type

• 500 bases
• 2500 bases
• 10 kbp
• 150 kbp
• 3 Mbp
• simple
• repeats
• BIG

• 1 locus EST,STS
• whole cDNA/EST
• gene, virus
• BAC, big virus
• bacterial genome
• YAC-size
• HUMAN, etc.

3
DNA Sequencing Methods

• Chain termination/Dideoxy/Sanger
• fluorescence paradigm, ABI, HOOD
• Sequencing by hybridization
• chips Affymetrix (Lander, et al)
• other formats
• Hyseq (Church, et al)
• Lark

4
Dideoxy/Chain Terminator/Sanger

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

5
Chain Terminator Basics
TGCA
Extend
dN ddN 100 1
Ladder n, n1...
6
Electrophoresis
7
Template Preparation

• ssDNA vectors
• M13
• pUC
• PCR
• dsDNA (/- PCR)

8
Primers

• Universal primers
• cheap, reliable, easy, fast, parallel
• BULK sequencing
• Custom primers
• expensive, slow, one-at-a-time
• ADAPTABLE

Primer Label
Dye Terminator
9
Extension Chemistry
100 termination Accurate Even signal

• Polymerase
• Sequenase
• Thermostable (Cycle Sequencing)
• Terminators
• Dye labels (Big Dye)
• spectrally different, high fluorescence
• (mass labels??)
• ddA,C,G,T with primer labels

10
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
11
Paradigm Instrument

• Applied Biosystems
• ABI3700 (early 1999)
• 1500 samples/day!
• http//www2.perkin-elmer.com/ga/3700/features.html
  
• ABI377 (gel) and ABI310 (capillary)

12
Alternate Instruments

• Molecular Dynamics, Beckman Coulter
• ALF, LiCor
• infrared detection

Not Complete List
13
Sample Output
14
Trace Editing

• EditView
• Mac
• Chromas
• WinNT
• Consed
• UNIX

15
Project Goals

• de novo sequence
• Chain terminators
• repetitive sequencing
• Sequencing by hybridization
• Chip technology, eg

16
Sequencing Strategies

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

Sequencing Assembly Finishing Annotation
Mix to Suit
17
Random Method

• Shear DNA (nebulize)
• finish ends, ligate into vector
• Produce template
• Sequence to target coverage
• read length (500 typical)
• accuracy (99 good)

Assemble Contigs
18
Random
19
Poisson Statistics
Lread length Nreads Ggenome size
P0e-L(N)/G
20
Poisson-2
Gap LengthP0G
21
Poisson-3
Gap NumberP0N (assume N500 bases)
22
4 Mbp Genome

• 10x Coverage
• 80,000 reads at 500 bases/read
• 4 gaps
• 400 bases in gaps

55 instrument days on ABI3700
23
3000 Mbp GenomeHUMAN
50000 instrument days on ABI3700
24
Automation
QT
25
Costs

• Raw cost 0.01/base
• Semi-finished 0.10 per base
• finished 0.30 per base
• High-quality Genome Project
• 0.50/base

26
Ordered Methods
Primer Walking
Nested Deletion
27
Limitations

• Slow, Expensive
• Expertise Needed
• especially nested deletion
• Repeat Problems
• especially primer walking

28
Finishing

• GOALS
• gt95 coverage on BOTH strands
• every base covered 3X
• resolve ambiguities
• Finish when random no longer productive (3-10 X
  range)

29
Finish-How

• Identify gaps, ambiguities
• Extend from end of contigs
• specific primers
• subclones, etc.
• Resolve ambiguities
• consensus or resequence
• specific primers, different chemistry

30
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)

31
Assembly Programs

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

32
Assembly ImprovementsRepeat Problems

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

33
Project Management

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

34
Annotation

• ORFs
• GRAIL, PowerBLAST
• Repeats
• Other Regions

Submit to Genbank ...HTGS (level1,2,3) ...nr
35
Sequencing by Hybridization
Hybridize labeled query DNA
CHIP OLIGOS (20-mers)
...gaactAatact... ...gaactCatact... ...gaactGatact
... ...gaactTatact...
site 1
...gaactaAtact... ...gaactaCtact... ...gaactaGtact
... ...gaactaTtact...
site 2
GAACTATGTACT
36
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
37
Physical MappingGenome Characterization

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