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Correlagen next gen presentation 042711
1. Sequencing
technologies
past, current and next generation
•Introduction to Helicos, Illumina and
Solid sequencing technology
•Applications
Robert Pinard
Some slides were adapted from
Karen Staehling-Hampton
The Stowers Genome Center
2. Introduction
• Genomics research has entered a new age, in
which deciphering the genome’s effect on
biology and medicine requires not only the
detection of mutations and sequence
variation, but also understanding the dynamic
nature of genome biology.
• The “Next Generation Sequencing” should be
called the Current Generation Sequencing
3. Overview of three major next
Generation Sequencing Technologies
• Illumina / GAII
• Helicos / Heliscope
• Life Tech / Solid
• (briefly Ion Torrent, PacBio
& Complete Genomics)
4.
5.
6. • The principle at the heart of all these
technologies is similar
• (sequence by synthesis)
Sanger Sequencing
9. • It is similar with the next generation
sequencers where the different platforms
either detect the incorporation of a
fluorescent nucleotide or a bi-product of the
reaction like the PPi or the release of a
proton by the DNA polymerase
10. Common Steps (to all platforms)
• Library Preparation
• Amplification Steps
• Attachment to a matrix (FlowCell)
• Sequencing & Detection
• Interpretation
11. Common Steps
• Library Preparation
• Amplification Steps
• Attachment to a matrix (FlowCell)
• Sequencing & Detection
• Interpretation
18. Step A
Region Selection: using Sure Select, capture region that we
really want to look at (complete exomes), subset of genes
(Familial Cardiac Genes).
19. Step B some PCR involved
• Pre-Hybrid Selection and post-Hybrid Selection
amplification PCR.
A- Pre-(to increase the pond of fragment DNA)
B- Post- (to increase the DNA that was captured)
20. Overall Steps
Modify ends/Adaptors Selection
Shear
Library Preparation
Amplify and or Attach
Amplification
Sequence & Detection
21. STEP C: Clonal amplification to increase signal detection
• EMULSION PCR or
CLUSTER AMPLIFICATION
Illumina
454/Roche
Solid/Life
Helicos
40. The addition of one of the four
454
deoxynucleotide triphosphates (dNTPs)(in the
case of dATP we add dATPαS which is not a
substrate for a luciferase) initiates the second
step. DNA polymerase incorporates the
correct, complementary dNTPs onto the
template. This incorporation releases
pyrophosphate (PPi) stoichiometrically.
ATP sulfurylase quantitatively converts PPi to
ATP in the presence of adenosine 5´
phosphosulfate. This ATP acts as fuel to the
luciferase-mediated conversion of luciferin to
oxyluciferin that generates visible light in
amounts that are proportional to the amount
of ATP. The light produced in the luciferase-
catalyzed reaction is detected by a camera and
analyzed in a program.
Unincorporated nucleotides and ATP are
degraded by the apyrase, and the reaction can
restart with another nucleotide.
53. Conclusions
• Several new platforms are emerging (variation
on a same theme) that will increase
throughput and reduce cost.
• The Next Gen Sequencing approaches are
really the Now Gen Sequencing approaches
and they are making a real impact in life
sciences and soon in clinical diagnostics
(starting with our own CGS test).
