1. High Throughput Sequencing Methods and Concepts
Cedric Notredame
adapted from S.M Brown

2. DNA Sequencing
The final essential tool in the molecular biology toolkit is the ability to read the base sequence of DNA molecules
Fred Sanger developed an elegant method to sequence DNA by using DNA polymerase enzyme
(for which he was awarded the Nobel Prize in 1980)
The Sanger method copies a piece of cloned DNA but some of the copies are halted at each base pair along the sequence.

5. Sanger Method
DNA polymerase adds free nucleotides to a primer which is complementary DNA template.
Sanger used some modified dideoxynucleotides to stop the replication process if they are incorporated in the growing DNA chain (terminators).
This produces a set of partial DNA copies of the original template sequence, each one stopping at a different base.
Sanger used 4 different reactions that each contained only terminators for one of the bases.
When the partial copies are sorted by size using electrophoresis, all fragment of a distinct size are terminated with the same base.

7. Automated Sequencing
Sequencing technology was improved in the late 1980s by Leroy Hood who developed fluorescent color labels for the 4 terminator nucleotide bases.
This allowed all 4 bases to be sequenced in a single reaction and sorted in a single gel lane.
Hood also pioneered direct data collection by computer.
Minor improvements in this technology now enable the sequencing of billion base genomes in a year or less.

9. Automated sequencing machines, particularly those made by PE Applied Biosystems, use 4 colors, so they can read all 4 bases at once.

11. DNA Sequencing capability has grown exponentially
DNA sequences in GenBank
Doubling time = 18 months

12. Next Generation Sequencing
454 Life Sciences/Roche
Genome Sequencer FLX: currently produces million bases per day per machine
Published 1 million bases of Neanderthal DNA in 2006
May 2007 published complete genome of James Watson (3.2 billion bases ~20x coverage)
Solexa/Illumina
10 GB per machine/week
May 2008 published complete genomes for 3 hapmap subjects (14x coverage)
ABI SOLID
20 GB per machine/week

13. “Paradigm Shift” Standard ABI “Sanger” sequencing
96 samples/day
Read length ~650 bp
Total = 450,000 bases of sequence data
454 was the game changer!
~400,000 different templates (reads)/day
Read length ~250 bp
Total = 100,000,000 bases of sequence data!!!

14. Solexa ups the Game Solexa (Illumina GA)
60,000,000 different sequence templates
(yes that is an insane 60 million reads)
36 bp read length
4 billion bases of DNA per run (3 days)

15. Nanotechnology
Each system works differently, but they are all based on a similar principals:
Shear target DNA into small pieces
bind individual DNA molecules to a solid surface,
amplify each molecule into a cluster
copy one base at a time and detect different signals for A, C, T, & G bases
requires very precise high-resolution imaging of tiny features
(Solexa has megapixels each)

16. One (of 800) tiles on Solexa Sequencer

17. Huge Amount of Image Data
The raw image data is truly huge: 1-2 TB for the Solexa, more for ABI-SOLID, less for 454
The images are immediately processed into intensity data (spots w/ location and brightness)
Intensity data is then processed into basecalls (A, C, T, or G plus a quality score for each)
Basecall data is on the order of 5-10 GB per run (or a week of runs for 454).

18. 454 First high-throughput DNA sequencer, commercially
available in 2004
Now (10/08) produces ~500 MB reads of 500 bp
Run of 8 samples in 10 hours, so can do multiple runs/week
Uses pyrosquencing, beads, and a microtiter plate
Low error rate, but insert/delete problems with homopolymers (stretches of a single base)

23. Illumina Genome Analyzer
Originally developed by Solexa, now subsidiary of Illumina.
Commercially available in 2006
Now produces 8-12 million reads per sample of 36 bp length = 10 GB/week.
Run takes 3 days for 7 samples.
Low error rate, mostly base changes, few indels

24. Illumina sequencing technology in 12 steps
Source: 24

25. 1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become double stranded
5. Denature the double- stranded molecules
6. Complete amplification
DNA
adapters
Randomly fragment genomic DNA and ligate adapters to both ends of the fragments

26. 4. Fragments become double stranded
adapter
DNA fragment
1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become double stranded
5. Denature the double- stranded molecules
6. Complete amplification
dense lawn of primers
adapter
Bind single-stranded fragments randomly to the inside surface of the flow cell channels

27. 1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become double stranded
5. Denature the double- stranded molecules
6. Complete amplification
Add unlabeled nucleotides and enzyme to initiate solid-phase bridge amplification

28. 4. Fragments become double stranded
1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become double stranded
5. Denature the double- stranded molecules
6. Complete amplification
Attached
terminus
free
Attached terminus
terminus
The enzyme incorporates nucleotides to build double-stranded bridges on the solid-phase substrate

29. 4. Fragments become double stranded
1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become double stranded
5. Denature the double- stranded molecules
6. Complete amplification
Attached
Attached
Denaturation leaves single-stranded templates anchored to the substrate

30. 4. Fragments become double stranded
1. Prepare genomic DNA
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become double stranded
5. Denature the double- stranded molecules
6. Complete amplification
Clusters
Several million dense clusters of double-stranded DNA are generated in each channel of the flow cell

31. 10. Image second chemistry cycle
7. Determine first base
8. Image first base
9. Determine second base
10. Image second chemistry cycle
11. Sequencing over multiple chemistry cycles
12. Align data
Laser
The first sequencing cycle begins by adding four labeled reversible terminators, primers, and DNA polymerase

32. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second chemistry cycle
11. Sequencing over multiple chemistry cycles
12. Align data
After laser excitation, the emitted fluorescence from each cluster is captured and the first base is identified

33. 10. Image second chemistry cycle
7. Determine first base
8. Image first base
9. Determine second base
10. Image second chemistry cycle
11. Sequencing over multiple chemistry cycles
12. Align data
Laser
The next cycle repeats the incorporation of four labeled reversible terminators, primers, and DNA polymerase

34. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second chemistry cycle
11. Sequencing over multiple chemistry cycles
12. Align data
After laser excitation the image is captured as before, and the identity of the second base is recorded.

35. 7. Determine first base
8. Image first base
9. Determine second base
10. Image second chemistry cycle
11. Sequencing over multiple chemistry cycles
12. Align data
The sequencing cycles are repeated to determine the sequence of bases in a fragment, one base at a time.

36. 10. Image second chemistry cycle
Reference sequence
7. Determine first base
8. Image first base
9. Determine second base
10. Image second chemistry cycle
11. Sequencing over multiple chemistry cycles
12. Align data
Unknown variant identified and called
Known SNP called
The data are aligned and compared to a reference, and sequencing differences are identified.

37. Illumina Genome Analyzer
Richard K. Wilson

38. Paired-End Sequencing
Nature Methods 5, May 2008

39. Sequencing Resynthesis of P5 Strand (15Cycles) Sequencing First ReadOH
Sequencing
First Read
Denaturation and
De-Protection OH
Denaturation and
Hybridization
P7 Linearization OH
Sequencing
Second Read
Denaturation and
Hybridization
Block with
ddNTPs
The steps up to and including the first read sequencing are pretty much the same as for a single read. The first read sequencing is where the single read protocol would stop. For the PE protocol, it continues with deprotecting the P5 primer using deprotection enzyme. Resynthesis of the P5 strand occurs over 15 cycles. P7 linearization uses Linearization 2 Enzyme. Blocking again occurs with ddNTPS and Blocking enzyme 1 and 2. Sequencing read 2 uses Read 2 PE Sequencing Primer.

41. ABI-SOLID First commercially available in late 2007
Currently capable of producing 20 GB of data per run (week)
Most users generate 6 GB/run
Reads ~30 bp long
Uses unique sequence-by-ligation method
“color-space” data
Very low error rate

44. Short Reads
Short reads from Nex-Gen machines are a challenge (Solexa = 36 bp)
Very hard to assemble whole genomes
Difficult to get any information on repeat regions
Requires many-fold coverage
New algorithms needed for many traditional bioinformatics operations
Reads are getting longer – another moving target

45. PacBio
High throughput Single Molecule Real Time (SMRT) Sequencing

46. PacBio
High throughput Single Molecule Real Time (SMRT) Sequencing

47. PacBio

48. PacBio

49. PacBio

50. Applications “If you build it, they will come.”
An explosion of scientific innovation!
Every new technology enables new applications, which are not directly foreseen by the original developers of the tech.
Cheap access to high-volume sequencing becomes a data collection method for many different types of experimental applications

51. When All You Have is a Hammer, All Problems Look Like Nails
Mark Twains

52. Applications