1. Canadian Bioinformatics Workshops

3. Introduction to next-gen sequencing
Informatics on High Throughput Sequencing Data
Introduction to next-gen sequencing
Francis Ouellette
July 25th 2008

4. Outline Sequencing DNA Next Generation Technologies
Solexa
SOLiD
454
Helicos
AB’s color space
What next, & things to keep in mind!

5. Adapted from John McPherson, OICR
Biological Research

6. History of DNA Sequencing
Adapted from Eric Green, NIH; Adapted from Messing & Llaca, PNAS (1998)
Avery: Proposes DNA as ‘Genetic Material’
Watson & Crick: Double Helix Structure of DNA
Holley: Sequences Yeast tRNAAla
1870
1953
1940
1965
1970
1977
1980
1990
2002
Miescher: Discovers DNA
Wu: Sequences  Cohesive End DNA
Sanger: Dideoxy Chain Termination
Gilbert: Chemical Degradation
Messing: M13 Cloning
Hood et al.: Partial Automation
Cycle Sequencing
Improved Sequencing Enzymes
Improved Fluorescent Detection Schemes
1986
Next Generation Sequencing
Improved enzymes and chemistry
Improved image processing
Efficiency
(bp/person/year) 1 15
150
1,500
15,000
25,000
50,000
200,000
50,000,000
100,000,000,000
2008

7. Basics of the “old” technology
Clone the DNA.
Generate a ladder of labeled (colored) molecules that are different by 1 nucleotide.
Separate mixture on some matrix.
Detect fluorochrome by laser.
Interpret peaks as string of DNA.
Strings are 500 to 1,000 letters long
1 machine generates 57,000 nucleotides/run
Assemble all strings into a genome.

8. Basics of the “new” technology
Get DNA.
Attach it to something.
Extend and amplify signal with some color scheme.
Detect fluorochrome by microscopy.
Interpret series of spots as short strings of DNA.
Strings are letters long
Multiple images are interpreted as 0.4 to 1.2 GB/run (1,200,000,000 letters/day).
Map or align strings to one or many genome.

9. From Debbie Nickerson, Department of Genome Sciences, University of Washington,

10. Differences between the various platforms:
Nanotechnology used.
Resolution of the image analysis.
Chemistry and enzymology.
Signal to noise detection in the software
Software/images/file size/pipeline
Cost $$$

11. Next Generation DNA Sequencing Technologies
Adapted from Richard Wilson, School of Medicine, Washington University, “Sequencing the Cancer Genome”
Next Generation DNA Sequencing Technologies
3 Gb ==

12. Solexa

13. Solexa-based Whole Genome Sequencing
Adapted from Richard Wilson, School of Medicine, Washington University, “Sequencing the Cancer Genome”

14. Solexa-based Whole Genome Sequencing
Adapted from Richard Wilson, School of Medicine, Washington University, “Sequencing the Cancer Genome”
Solexa flow cell
~50M clusters are sequenced per flow cell.

15. Debbie Nickerson, Department of Genome Sciences, University of Washington, http://tinyurl.com/6zbzh4

16. 454

17. Roche / 454 : GS FLX Real Time Sequencing by Synthesis
Chemiluminescence detection in pico titer plates
Amplification: emulsion PCR
Pyrosequencing
up to 400,000 reads / run
on average 250 bases / read (and longer)
up to 100 Mb / run

18. Roche / 454 : GS FLX Made for de novo sequencing.
Too expensive for resequencing.
For example, this platform will be used a lot by laboratories doing new bacterial genomes.
Baylor Genome Center involved in Sea Urchin, Bee, Platypus genomes: They have a number of 454.

19. Helicos

20. Single Molecule Sequencing
Adapted from: Barak Cohen, Washington University, Bio
Single Molecule Sequencing
Microscope slide * * *
Single DNA
molecule
Super-cooled
TIRF microscope
primer
dNTP-Cy3 *
Helicos Biosciences Corp.

21. Helicos Approximate Data Production per Run at Current Peak Throughput (1 strand/µ2)
Single Pass Dual Pass
7 day run 14 day run
Image Data: TB 60 TB
Diagnostic Images: 350 GB 600 GB
Object Table: TB 6 TB
Sequence Data: GB GB
Log Files: GB 600 GB
Total ~4.5 TB ~7.8 TB (w/o full image stack)

22. ABI SOLiD

24. File management

25. SOLiD color space

39. It’s more complicated! Get files with quality scores
Get files with miss-matches
Need to align them to a reference genome
Multiple tools do this today … and there will be more later.
What do you do? Do it all!

40. Things to keep in mind
All people are learning, if you don’t know, ask, and they probably won’t know either, and you can figure it out together!
The technology is changing – This workshop next year will be totally different!
We can only do so much in two days – you will need to find things, find people who can help you, and you will need to teach your friends!

41. Other factors Changing technology Changing price structure
New and disappearing companies?
Changing price structure
Cost of machine
Cost of operation (reagents/people)
Service from the company
1 machine vs (2 or 3 machines) vs 40 machines.
Changing software and processing

42. Pacific Biosystems (PacBio)

43. Questions?
Coffee break!

44. Day 1