Fu-Sequencing platforms and applications 2014 [兼容模式]
Transcript of Fu-Sequencing platforms and applications 2014 [兼容模式]
Next-Gen High Throughput Sequencing Technologies and Applications in Basic andTechnologies and Applications in Basic and
Translational Research
Xiang-Dong Fu
Wuhan University/UC San DiegoWuhan University/UC, San Diego
High Throughput Sequencing: Capacity
ER Mardis. Nature 470, 198-203 (2011)
OutlineOutline
• 454: Pyrosequencing
Sequencing Technologies454: Pyrosequencing
• SOLiD: Sequencing by ligation• Ion Torrent: High Throughput pH Array• Solexa/Illumina: Sequencing by SynthesisSolexa/Illumina: Sequencing by Synthesis• PacBio: Real-time imaging • Future sequencing technologies
•Fluorescent in situ sequencing (FISSQ)q g ( )•FRET-based Nanomachine
Applications• De novo and target-specific sequencing• RNA-seq for transcriptome analysis• ChIP-seq for protein-DNA interactionsq p• CLIP-seq for protein-RNA interactions• GRO-seq for detecting nascent RNA• Ribosomal profiling for translating mRNA
454 Pyrosequencing (Roche)-1
A. Library construction
B. Emulsion PCR
454 Pyrosequencing (Roche)-2C. Formation of bead array
D. Sequencing reaction
SOLiD: Sequencing By Ligation-1
SOLiD: Sequencing By Ligation-2
SOLiD: Sequencing By Ligation-3(each base is decoded twice)(each base is decoded twice)
Ion Torrent (Life Technologies)-1(microarray of pH meters)
Ion Torrent (Life Technologies)-2(sequencing by synthesis)(sequencing by synthesis)
PacBio: Real Time Sequencing by Synthesis
Sequencing by SynthesisSequencing by Synthesis
The Illumina Success
Solexa/Illumina Sequencing-1(generation of single molecule clusters)(generation of single molecule clusters)
Solexa/Illumina Sequencing-2(sequencing by synthesis)(sequencing by synthesis)
Power House of Next-Gen SequencingIllumina Overview
Future Sequencing PlatformsFuture Sequencing Platforms
Fluorescent In Situ Sequencing• Single cell and single molecule resolution• Single cell and single molecule resolution• Subcellular distribution of different RNA• Mutational detections in individual cellsutat o a detect o s d dua ce s
Sequencing in real timeq g•No amplification needed•Each Qdot as a sequencing machine
Future SequencingSequencing
Technology-1: FluorescentFluorescent
In Situ S iSequencing
(FISSQ)
Lee et al., Science 343:1360,, 2014
Future Sequencing Technology-2: Fluorescent In Situ Sequencing (FISSQ)Fluorescent In Situ Sequencing (FISSQ)
Hybridization signals on mouse brain
12
14
6
8
10
12
RFU
Signal/Background
Abs Intensity
0
2
4
6R0
0 10 20 30 40 50 60
Re-probing cycles
Future Sequencing Technology-3: Fl I Si S i (FISSQ)Fluorescent In Situ Sequencing (FISSQ)
Lee et al., Science 343:1360-1363, 2014
Single Molecule Real-Time DNA Sequencing-1g q g
Qdot-labeled DNA Polymerase y
Single Molecule Real-Time DNA Sequencing-2g q g
Fluorescence Resonance Energy Transfer (FRET) from Qdot to incoming DNA synthesis substrate g y
Single Molecule Real-Time DNA Sequencing-3g q gRecoding sequences in real time series
Single Molecule Real-Time DNA Sequencing-4What the data look like
Applications of Next-Gen Sequencing
D d ifi i• De novo and target-specific sequencing• RNA-seq for transcriptome analysis• ChIP-seq for protein-DNA interactions• ChIP-seq for protein-DNA interactions• CLIP-seq for protein-RNA interactions• GRO-seq for detecting nascent RNAq g• Ribosomal profiling for translating mRNA
RNA-seq: Focusing on the 3’ endRNA seq: Focusing on the 3 end
A SAGE like strategy B Random priming based strategyA. SAGE-like strategy B. Random priming-based strategy
Cap (A)n Cap (A)ndTdTn
cDNA conversiondTn
cDNA conversion
R d i i
dTn
Restriction digestion
5’ Linker ligatiion xxxxxxxx
Random priming
dTn
Type II restrictiondigestion dTn
Elution
3’ Linker ligatiionPCR
High Throughput Sequencing High Throughput Sequencing
RNA tags at the 3’ end of transcripts detected by MASPs
A B
)
detected by MASPs
p 2
(tag
coun
ts)
CExp 1 (tag counts)
Exp
C
DD
Detection of DNA Binding Events (ChIP-seq)
A ib d I l d TFAntibody Isolated TFBinding sites
TF TF
Chromatin Immunoprecipitation
(ChIP)linker
High ThroughputSequencing
( )
Genomic Array
Target DNA
q g(Seq)
Genomic Array
Crosslinking agents: UV, formaldehyde, Glutarldehyde
Detection of RNA Binding Events: (CLIP-seq)
Yeo et al., NSMB 16:130-170, 2009 Konig et al., NSMB 17:909-915, 2010
microRNA Target Identification by Crosslinking, Ligation and Sequencing of HybridsLigation and Sequencing of Hybrids
RISC 3’OH RISC5’P
3’OH 3’P
RNase A+T
RISC RISC
PPase
RISC
3’P
3’OH5’OH
RISC
3’OH
Kinase
RISC 5’ linker
3’ linker additionPK5’ Linker addition
RISC5’P
3’OH
3’P
5 linker
3’ linker
Deep Sequencing3 P
Kudla et al., PNAS 108:1001-10015, 2011Helwark et al., Cell 153:654-665, 2013
Selections and Deep Sequencingto Determine RNA Binding Specificityto Determine RNA Binding Specificity
Campbell et al., Cell Rep 1:570-581, 2012
Ribosomal Profilingg
Ingolia et al., Science 234:218-223, 2009
Detection of Nascent RNA by Global Nuclear Run On Coupled with Deep SequencingRun-On Coupled with Deep Sequencing
Poly A addition at the 3’ endand RT primer extension
Pol II complex Nascent transcripts
AAAAAAdT-
RNAcDNA
dT
CircularizationSarkosyl + NTPs + Br-UTP ( )
Pause sites
dT-
Cleavage and PCRAnti BrU affinity purificationMagnetic beads
dT-
High throughput sequencing
Anti-BrU affinity purificationMagnetic beads
dT-
g g p q g
Core et al., Science 322:1846-1848, 2008Core et al., Science 322:1845-1848, 2008Lin et al., NSMB 15:819-826, 2008
Time Course GRO-seq to Detect Genome-wide Translation Elongation RateTranslation Elongation Rate
Yu and Fu, Unpublished Result