新一代测序技术solaxe+solid+454

A
C 1st Base
G T
Sequencing Overview SOLID Example I:
• Color signals translates to 4 possible answers • Across the 4 possible answers, each position has a unique base • If you know ONE base in the sequence, you can determine the sequence.
Sequencing By Synthesis Load beads into PicoTiter™ Plate Sequencing by synthesis Photons Generated are Captured by Camera Sequencing Image Created
454 Sequencing: BaseCalling
• Count the photons generated for each “flow” • Base call using signal thresholds • Delivery of one nucleotide per flow ensures accurate base calling
Emulsion PCR Amplification
Anneal sstDNA to an excess of DNA capture beads Emulsify beads and PCR reagents in water-in-oil microreactors Clonal amplification occurs inside microreactors
0
Dye XY1 XY2 XY3 XY4 FAM AA CC GG TT
1
Cy3 AC CA GT TG
2
TXR AG GA CT TC
3
Cy5 AT TA CG GC
Sequencing Overview SOLID
A 1st Base
2nd Base C G T
Example II:
A
C
G T
Large Polystyrene Bead
P2
P1
Centrifuge using a Glycerol Gradient
Captured beads (+ templates) in supernatant
Uncaptured beads (no template) in pellet
SOLiD: 4-color, 2-base encoding
Flow Order 4-mer
T A C G
Measures the presence or absence of each nucleotide at any given position
3-mer
KEY (TCAG)
2-mer
1-mer
Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )
Genome fragmented by nebulization No cloning; no colony picking sstDNA library created with adapters A/B fragments selected using avidin-biotin purification
AB /SOLID Workflow
DNA Library Preparation emPCR Sequencing
DNA Library Preparation Genome fragmented by nebulization Adaptor ligation
Emulsion PCR Amplification
42
NTP PPi PPi + APS ATP ATP + Luciferin
Sequencing Overview GS Flx (454)
luciferase Oxyluciferin + Light
Sequencing Overview GS Flx (454)
1. DNA library preparation
Sequencing Overview GS Flx (454)
3.Loading DNA Beads into the PicoTiter™Plate
Load beads into Pico Titer ™ Plate
Well diameter: average of 44 µm
Load Enzyme beads
3’ N N N G GsZ Z Z
3’ N N N G TsZ Z Z
3’ N N N C TsZ Z Z
3’ N N N C GsZ Z Z
3’ N N N T TsZ Z Z
3’ N N N T GsZ Z Z
3’ N N N T CsZ Z Z
3’ N N N G CsZ Z Z
Following base
•Start Sequencing • Grow Clusters Clusters • Grow •Start Sequencing •Cluster Density • 5 hours •<30 min. hands-on •3 days single-read Evaluation •Or split process time,5 hours run •4 images per tile in stages •10 days pairedper cycle •Safe stopping end run •Run time: 2-3 points (2*75 bases) days
Clonal amplification Break microreactors, occurs inside enrich for DNAmicroreactors positive beads
sstDNA library
Clonally-amplified sstDNA attached to bead
3’ N N N A AsZ Z Z 3’ N N N C CsZ Z Z 3’ N N N A CsZ Z Z 3’ N N N C AsZ Z Z 3’ N N N A GsZ Z Z 3’ N N N G AsZ Z Z 3’ N N N A TsZ Z Z 3’ N N N T AsZ Z Z
Sequencing Overview GS Flx (454)
5.Sequencing And Image Capture
The sequencing instrument consists of the following major subsystems: (a) a fluidic assembly ， (b) a flow chamber that includes the wellcontaining fibre-optic slide， (c) a CCD camerabased imaging assembly， and a computer that provides the necessary user interface and instrument control.
•Firecrest: Image Analysis •Bustard: Basecalling •Gerald: Sequence Alignment
Sequencing Overview / Solexa
Sequencing Overview / Solexa
Sequencing Overview / Solexa
Roche /454 GS FLX Workflow
DNA Library Preparation emPCR Sequencing
DNA Library Preparation Genome fragmented by nebulization Adaptor ligation sstDNA library created with adapters A/B fragments selected using avidin-biotin purification
gDNA
sstDNA library
Sequencing Overview GS Flx (454)
2.Emulsion Based Clonal Amplification
Anneal sstDNA to an excess of DNA capture beads
Emulsify beads and PCR reagents in water-in-oil microreactors
AA CC GG TT
AC CA GT TG
AC CA GT TG
AA CC GG TT
AG CT GA TC
AT CG GC TA
AA CC GG TT
AG CT GA TC
AG CT GA TC
If know first base is an A then immediately it decodes 2nd base. This must be an A as Blue translates 2nd base A if first base A
0 Dye XY1 XY2 FAM AA CC
1 Cy3 AC CA
2 TXR AG GA
3 Cy5 AT TA
Leading base A C G T
A
C
G
T
XY3
XY4
GG
TT
wenku.baidu.com
GT
TG
CT
TC
CG
GC
Sequencing Overview SOLID
Consequences of 2 Base Pair Encoding
Sequencing by Synthesis Beads attached to glass surface in a random array Sequencing by synthesis Photons Generated are Captured by Camera Sequencing Image Created
P2
P1
P2 Beads with no product Beads with amplified product (~40K PCR products per bead)
Sequencing Overview SOLID
3. Enrichment
P2 enrichment oligo P1 P2
Illumina/GA Workflow
Sample Prep Sample Prepare •Genomic •mRNA •Small RNA •ChIP-Seq Cluster ClusterStation Station Genome 1G Analyzer Analyzer Analysis Pipeline
Sequencing Overview / Solexa
Sequencing Overview / Solexa
Sequencing Overview / Solexa
SBS（sequencing-by-synthesis ）
Sequencing Overview / Solexa
Sequencing Overview / Solexa
11
Mix PCR aqueous phase into a water-in-oil emulsion and carry out emulsion PCR
Sequencing Overview SOLID
Beads collected following emulsion PCR:
2nd Base
A
C
G
T
• Detecting a single color does not indicate a base • Each reading contains information from two bases • To decode the bases you must know one of the bases in the sequence