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