1. Basic Techniques Project Design Process Improvements
DNA Sequencing
Basic Techniques
Project Design
Process Improvements
9/18/2018
Chuck Staben

2. Project Size/Type 500 bases 2500 bases 10 kbp 150 kbp 3 Mbp BIG
simple
repeats
BIG
1 locus EST,STS
whole cDNA/EST
gene, virus
BAC, big virus
bacterial genome
YAC-size
HUMAN, etc.
9/18/2018
Chuck Staben

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
9/18/2018
Chuck Staben

4. Dideoxy/Chain Terminator/Sanger
Template
Primer
Extension Chemistry
polymerase
termination
labeling
Separation
Detection
9/18/2018
Chuck Staben

5. Chain Terminator Basics
Template-Primer
Target
TGCA
ddA
ddG
ddC
ddT
Terminators
Extend
ddA A
ddC
dN : ddN
100 : 1
Ladder
n, n+1... AC
ddG
ACG
ddT
9/18/2018
Chuck Staben

6. Electrophoresis
9/18/2018
Chuck Staben

7. Template Preparation ssDNA vectors PCR dsDNA (+/- PCR) M13 pUC
9/18/2018
Chuck Staben

8. Primers Primer Label Dye Terminator Universal primers Custom primers
cheap, reliable, easy, fast, parallel
BULK sequencing
Custom primers
expensive, slow, one-at-a-time
ADAPTABLE
Primer Label
Dye Terminator
9/18/2018
Chuck Staben

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
9/18/2018
Chuck Staben

10. Separation migration ~1/log N 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
9/18/2018
Chuck Staben

11. Paradigm Instrument Applied Biosystems ABI3700 (early 1999)
1500 samples/day!
ABI377 (gel) and ABI310 (capillary)
9/18/2018
Chuck Staben

12. Alternate Instruments
Molecular Dynamics, Beckman Coulter…
ALF, LiCor
infrared detection
Not Complete List
9/18/2018
Chuck Staben

13. Sample Output
1 lane
9/18/2018
Chuck Staben

14. Trace Editing EditView Chromas Consed Mac WinNT UNIX 9/18/2018
Chuck Staben

15. Project Goals de novo sequence repetitive sequencing Chain terminators
Sequencing by hybridization
Chip technology, eg
9/18/2018
Chuck Staben

16. Sequencing Strategies
Random Sequence
Brute Force
Ordered
Divide and Conquer
Sequencing
Assembly
Finishing
Annotation
Mix to Suit
9/18/2018
Chuck Staben

17. Random Method Assemble Contigs Shear DNA (nebulize) Produce template
finish ends, ligate into vector
Produce template
Sequence to target coverage
read length (500 typical)
accuracy (99% good)
Assemble Contigs
9/18/2018
Chuck Staben

18. Random T C No coverage DISAGREEMENT Only 1 strand 9/18/2018
Chuck Staben

19. Poisson Statistics P0=e-L(N)/G L=read length N=#reads G=genome size
9/18/2018
Chuck Staben

20. Poisson-2
Gap Length=P0G
9/18/2018
Chuck Staben

21. Gap Number=P0N (assume N=500 bases)
Poisson-3
Gap Number=P0N (assume N=500 bases)
9/18/2018
Chuck Staben

22. 4 Mbp Genome 55 instrument days on ABI3700 10x Coverage
80,000 reads at 500 bases/read
4 gaps
400 bases in gaps
55 instrument days on ABI3700
9/18/2018
Chuck Staben

23. 3000 Mbp Genome HUMAN 50000 instrument days on ABI3700 300 machines,
3 years
Plenty
9/18/2018
Chuck Staben

24. Automation QT
9/18/2018
Chuck Staben

25. Costs $0.50/base Raw cost ~$0.01/base “Semi-finished” $0.10 per base
High-quality Genome Project
$0.50/base
9/18/2018
Chuck Staben

26. Ordered Methods
Primer Walking
Nested Deletion
9/18/2018
Chuck Staben

27. Limitations Slow, Expensive Expertise Needed Repeat Problems
especially nested deletion
Repeat Problems
especially primer walking
9/18/2018
Chuck Staben

28. Finishing GOALS Finish when random no longer productive (3-10 X range)
>95% coverage on BOTH strands
every base covered 3X
resolve ambiguities
Finish when random no longer productive (3-10 X range)
9/18/2018
Chuck Staben

29. Finish-How Identify gaps, ambiguities Extend from end of contigs
specific primers
subclones, etc.
Resolve ambiguities
consensus or resequence
specific primers, different chemistry
9/18/2018
Chuck Staben

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)
9/18/2018
Chuck Staben

31. Assembly Programs PHRAP FAMILY SeqWeb…. phrap, kangaroo, phrapo,
GAP4, TIGRAssembler,...
GCG
gelstart, gelenter, gelmerge, gelassemble, geldisassemble
thinly veiled vi editor
SeqWeb….
9/18/2018
Chuck Staben

32. Assembly Improvements Repeat Problems
Multiple fragment sizes in 1 project
Use length/distance info
9/18/2018
Chuck Staben

33. Project Management Editing and Assembly Databases RepeatMasker
Phred/Phrap
Consed
Databases
ACeDB
A C. elegans database
Oracle
9/18/2018
Chuck Staben

34. Annotation Submit to Genbank ...HTGS (level1,2,3) ...nr ORFs Repeats
GRAIL, PowerBLAST
Repeats
Other Regions
Submit to Genbank
...HTGS (level1,2,3)
...nr
9/18/2018
Chuck Staben

35. Sequencing by Hybridization
Hybridize
labeled query DNA
CHIP OLIGOS (20-mers)
...gaactAatact...
...gaactCatact...
...gaactGatact...
...gaactTatact... A C G T
site 1
...gaactaAtact...
...gaactaCtact...
...gaactaGtact...
...gaactaTtact...
site 2
GAACTATGTACT
9/18/2018
Chuck Staben

36. Modern Sequencing Challenges
Heterozygous DNAs
germline differences
somatic variation
Massive sequencing
population studies
genome scans
Minimal sample preparation
“Doctor’s Office”
Chips,
Quantitative Seq
Automation
Miniaturization
9/18/2018
Chuck Staben

37. Physical Mapping Genome Characterization
Genome fragmentation and cloning
vectors, etc.
Physical map assembly
hybridization
fingerprinting
9/18/2018
Chuck Staben