1

SMRT Cell

Template Prep &Binding Kit

Sequencing Kit

PacBio RS ---

GC

Gene Express

2012 8 24-26

2012

2012 8 24-26

2012 10 19-21

2012 10 19-21

1

PCR PCR qPCR

Bioline

Bioline

Enzymatics

Amnis Im-

Enzymatics

ISO1-3485:2003 9001:2000 FDA 21CFR.

820

ageStreamX Mark II。ImageStreamX Mark II ImageStream

ImageStream

ImageStreamX Mark II

ImageStreamX Mark II

Mark II

Mark II

uL

Mark II

2012 10 12-16

2012 10

200

Illumina NūPCR

NūPCR

qPCR qPCR

NūZyme

NūPCR

PCR

DesignStudio

NuPCR

™

™

Gene Express

2

CELL

NGS

CELL

SNP CNV

GWAS

SNP SNP

CELL

CELL

CELL

NGS

Peng Jin, Bing Ren, Chuan He et al, Base-Resolution Analysis

of 5-Hydroxymethylcytosine in the Mammalian Genome, Cell

149, 1368 - 1380, June 8, 2012

Bisulfite Sequencing

5mC 5mC 5hmC

5mC

5hmC

5hmC 5hmC

sequencing（TAB-Seq）

5hmC 5hmC 5mC

5hmC

（sequence bias）

strand asymmetry 5hmC

5hmC

CELL Tet-assisted bisulfite

NGS

Tet bisulfite

NGS

Cathy L. Barr, Bing Ren et al, Base-Resolution Analyses of Sequence

and Parent-of-Origin Dependent DNA Methylation in the Mouse

Genome, Cell 148, 816–831, February 17, 2012

DNA

MethylC-Seq，ChIP-Seq， RNA-Seq

SNP

Christopher E. Mason, Samie R. Jaffrey et al, Comprehensive

Analysis of mRNA Methylation Reveals Enrichment in 3 UTRs and

near Stop Codons, Cell 150, 1–12, July 6, 2012

(m6-

A)。 MeRIP-Seq

m6A RNA NGS

RNA-seq

RNA

NGS

m6A RNA

NGS RNA

cDNA

Gene Express

3

DNA A total of 183,916

somatically acquired base substitutions were identified. In

protein coding regions, there were 1,372 missense, 117

nonsense, 2 stoplost, 37 essential splice-site, and 521 silent

mutations. Of the 2,869 indels identified, 2,233 were deletions,

544 insertions and 92 complex. There were 21 coding indels, of

which 15 were predicted to result in a translational frameshift

and six were in-frame. In addition, 1,192 structural variants

(rearrangements), 16 homozygous deletions, and 14 regions of

increased copy number （amplifications） were identified.

SNP

CNV

NGS

DNA SNP、CNV

SNP CNV

Michael R. Stratton1, the Breast Cancer Working Group of the

International Cancer Genome Consortium et al， Mutational

Processes Molding the Genomes of 21 Breast Cancers， Cell

149, 979–993, May 25, 2012

Peter J. Campbell, Breast Cancer Working Group ofthe

International Cancer Genome Consortium et al, The Life

History of 21 BreastCancers, Cell 149, 994–1007, May 25,

2012

DNA

Jian Wang, Yingrui Li, Xiuqing Zhang et al, Single-Cell Exome

Sequencing and Monoclonal Evolution of a JAK2-Negative

Myeloproliferative Neoplasm, Cell 148, 873–885, March 2,

2012

Jian Wang, Michael Dean, Yingrui Li et al, Single-Cell Exome

S e q u e n c i n g R e v e a l s S i n g l e - N u c l e o t i d e M u t a t i o n

Characteristics of a Kidney Tumor, Cell 148, 886–895, March

2, 2012

DNA NGS

James F. Gusella et al, Sequencing Chromosomal Abnormalities

Reveals Neurodevelopmental Loci that Confer Risk across Diagnostic

Boundaries, Cell 149, 525–537, April 27, 2012

Balanced chromosomal abnormalities

Li-SH-

Peter Lichter,Stefan M. Pfister,Jan O. Korbe,et al,Genome、

Sequencing of Pediatric Medulloblastoma Links Catastrophic

DNA Rearrangements with TP53 Mutations, Cell 148, 59–71,

January 20, 2012

NGS SNP

chromothripsis

Fraumeni syndrome Sonic-Hedgehog medulloblastoma

H-MB

TP53

SHH-MBs TP53

chromothripsis AML

Cell 144, 27–40 NGS SNP

chromothripsis

Gene Express

4

the abundance of alternative spliced isoforms,

heteroallelic expression, RNA edits, expression of miRNAs。

blood components (peripheral blood mononuclear

cells [PBMCs], plasma and sera)

Our analysis detected many

single nucleotide variants (SNVs), small insertions and

deletions (indels) and structural variants (SVs; large insertions,

deletions, and inversions relative to hg19). 134,341 (4.1%)

high-confidence SNVs are not present in dbSNP, indicating

that they are very rare or private to the subject. Only 302

high-confidence indels reside within RefSeq protein coding

exons and exhibit enrichments in multiples of three nucleotides

(p < 0.0001). In addition to indels, 2,566 high-confidence SVs

were identified and 8,646 mobile element insertions were

identified.

MHC/HLA

16sRNA

RNA CELL NGS

RNA

RNA NGS

NGS SNP

Sanger Taqman

SNP、CNV Taqman

(BCAs)。BCA inversions,excision/insertions,translocations

BCA

NGS BCA

BCA

BCA

NGS

BCA (Am. J. Hum. Genet. 88,

B (Cell 147, 107–119;

147,95–106)、 (Cell 144, 27–40)、

(Nat. Genet. 44,

390–397)

NGS

Libraries were created by four different methods

optimized for delineating BCAs, including (1) standard insert

paired-end sequencing，(2) mate-pair sequencing (long

2,000–4,000 bp inserts)， (3) customized jumping libraries

(long 3,000–4,500 bp inserts)， (4) capture of breakpoints

method (CapBP) for rearrangements previously localized.

Austin Smith, HendrikG. Stunnenberg et al, The Transcriptional and

Epigenomic Foundations of Ground State Pluripotency, Cell 149,

590–604, April 27, 2012

（RNA-seq）

(ChIP-seq)

Cell, Volume 149, Issue 2, 467-482, 13 April 2012一

RNA-seq ChIP-seq T

Michael Snyder et al, Personal Omics Profiling Reveals

Dynamic Molecular and Medical Phenotypes, Cell 148,

1293–1307, March 16, 2012

Cell

Gene Express

5

Gene Express

6

PacBio

PacBio RS DNA

Steven Turner Jonas

Korlach DNA

DNA

15nm

DNA

《

Science》 Korlach

Turner Pacific Biosciences PacBio

PacBio RS(

Pacific Biosciences PacBio RS

（Single Molecule Real

Time, SMRT）DNA SMRT Cell

DNA DNA

DNA PacBio

RS

NGS

NGS

PCR

PacBio RS

NGS

PacBio RS

DNA

DNA

DNA链

DNA 15nm

DNA DNA

PacBio RS

DNA DNA

DNA

DNAPacBio RS

DNA

DNA DNA

PacBio RS SMRT Cell

Cell

ZMW(zero-mode waveguide

ZMW ZMW

DNA DNA

ZMW

DNA

DNA DNA

ZMW

DNA DNA ZMW

PacBio Magbead

DNA -DNA ZMW

Gene Express

7

Magbead

PacBio RS ZMW PacBio

PacBio RS

PacBio

PacBio RS DNA

IPD

SMRT Cell

DNA

DNA

DNA

PacBio RS

ZMW 600nm ZMW

ZMW

30nm ZMW

DNA

ZMW

PacBio RS

ZMW

ACGT

PacBio

IPD

DNA DNA

DNA DNA

IPD

DNA IPD DNA IPD IPD

IPD

mA、mC hmC Nature Method

DNA 4-mC、6-mA、

5-mC和5-hmC

DNA

DNA

SMRT

DNA

Gene Express

8

PacBio RS SMRT

PacBio RS

SMRT Cell

RS Touch

SMRT Cell

QV

PacBio RSPacbio

PacBio RS SMRT

SMRT bell

DNA

CCS

CLR

SMRT

A

B

C

PacBio

SMRT Pipe、SMRT Portal SMRT View

SMRT Portal

SMRT View

SNP

RS Remote

NGS

3-10kb

SMRT

Bell

PacBio

Gene Express

9

NGS

GC

PacBio RS

NGS

PacBio RS

NGS PacBio

C2

PacBio RS NGS

PCR

GC

A+T CG

PacBio C2 C1

C2

SMRT Cell

PacBio

IPD

IPD 4mC 6mA

IPD

PacBio

SNP

SNP

1. Roberts R.J., Vincze T., Posfai J. and Macelis D. REBASE--a

database for DNA restriction and modification: enzymes, genes

and genomes. Nucleic Acids Res, 38, D234-236 (2010).

2. Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC,

Clark TA, Korlach J, Turner SWDirect detection of DNA

methylation during single-molecule, real-time sequencing.

Nature Methods 7:461-465. (2010)

3. Song CX, Clark TA, Lu XY, Kislyuk A, Dai Q, Turner SW, He

C, Korlach J.Sensitive and specific single-molecule sequencing

of 5-hydroxymethylcytosine. Nat Methods. 2011 Nov

20;9(1):75-7

Gene Express

10

RNA

ChIP

AFA

DNA

DNA

20kHz～25kHz

100mm

DNA

Covaris高性

AFA

Covaris AFA

M、S、E、L

DNA/RNA/

Covaris

Covaris Adaptive Focused Acoustics

AFA

AFA

Gene Express

11

Covaris AFA

m/sec AFA

0.5MHz 1mm

AFA

AFA

80bp-5Kb

AFA

g-TUBE™Agilent 2100

12K

AFA

80bp-5Kb，g-TUBE 6Kb-

20Kb

truChIP

20Kb

µg µL

Covaris g-TUBE™ 6Kb-

Covaris truChIP™

truChIP™

Covaris AFA

CryoPrep ChIP-seq™

Gene Express

12

Covaris ChIP-seq

Gene Express

13

SNPs

Affymetrix

Affymetrix

SNP CytoScan HD CytoScan HD

750k

SNP6.0

SNP

CNV

SNP

SNP

LOH

FISH CGH

SNP-CNV SNP

SNP

CytoScan HD

marker

marker

750k

SNP

ISCA OMIM

Morbid OMIM Morbid

Affymetrix

Light-Controlled In Situ Synthesis

of DNA Microarrays

Affymetrix

Affymetrix

Nature、Science、Cell

Affymetrix

CytoScan HD

Affymetrix Frank Witney

Affymetrix CytoScan HD

Witney

CytoScan HD芯

SNP

Affymetrix ——Chromosome

Analysis Suite(ChAS)

excel pdf

Gene Express

14

SNP6.0 CytoScan HD

Guangyu Gu

Affymetrix CytoScan HD

10q24.32

670kb TMEM180,

ACTR1A,SUFU,TRIM8,ARL3,SFXN2,C10orf26,CYP17A1,

C10orf32,AS3MT,CNNM2

24.1 SUFU TRIM8

SUFU TRIM

Genomic Variants

10q24.32

SUFU,TRIM8

10q

SNP

LOH DNA

FISH

FISH

FISH

Affymetrix —CytoScan HD

FISH

Todd Christensen

James Tepperberg

Affymetrix SNP

Affymetrix SNP CytoScan HD

CGH

Hiba

HD

697k

Affymetrix CytoScan

HD 19p13.2

697k

DNM2(Charcot Marie Tooth Axonaltype 2M LDLR(Autosomal

Dominant Familial Hypercholesterolemia LDLR

DNA

LDLR

Risheg CytoScan

Gene Express

15

Yiping

Shen

FISH

and Speech Delay Hiba Risheg1, Sarah Parisotto2, Brooke

Rush3, Helio Pedro2, Peter Papenhausen3, Elisabeth Keitges1

1Cytogenetics, Laboratory Corporation of America, Seattle,

WA, 2Hackensack University Medical Center, Hackensack, NJ,

3Cytogenetics, Laboratory Corporation of America, Research

Triangle Park, NC American College of Medical Genetics

(ACMG) 2012, 30 March 2012, Abst #144.

3. Utilization of magnetic activated cell sorting and high

resolution SNP microarrays improves diagnostic yield and

prognostic value in clinical testing for patients diagnosed with

multiple myeloma. Todd Christensen1, Weiwen Deng1, Bonnie

McMahill1, Joseph Schappert1, Weihua Liu1, Reza Saleki1,

Ying Zou1 1Pathology Associates Medical Laboratories,

Spokane, WA, United States American College of Medical

Genetics (ACMG) 2012, 30 March 2012, Abst #142.

4. The Advantage of SNP Microarray Compared to

Chromosome Analysis in the Evaluation of POC for Fetal

Demise James Tepperberg1, Holly Taylor1, Rachel Burnside1,

Brooke Rush1, Inder Gadi1, Vikram Jaswaney1, Elisabeth

Keitges2, Romela Pasion1, Karen Phillips1, Venktswara

Potluri3, Hiba Risheg2, Stuart Schwartz1, Janice Smith3, Peter

Papenhausen11Cytogenetics,LabCorp,RTP,NC, 2Cytogenetics,

LabCorp, Seattle, WA, 3Cytogenetics, LabCorp, Houston, TX

American College of Medical Genetics (ACMG) 2012, 29

March 2012, Abst #151.

5. Standardization and Diversification of Copy Number

Microarray Testing for Clinical Diagnostics—Implications of

the Cross-Platform/Algorithm Study on Clinical Diagnostic

Chromosomal Microarray Analysis Yiping Shen, Yu An, and

Bai-Lin Wu Clin. Chem., Oct 2011; 57: 1354 - 1356.

X

1. Genomic microarray analysis of chronic lymphocytic leukemia

reveals a recurrent monoallelic deletion of 10q24.32；

Guangyu Gu1,2, Maria Sederberg2, Sarah T South1,2 1Depts

of Pediatrics and Pathology, University of Utah, Salt Lake City,

UT, 2ARUP Institute for Clinical & Experimental Pathology

ARUP Laboratories, Salt Lake City, UT American College of

Medical Genetics (ACMG) 2012, 29 March 2012, Abst #139.

2. Two Year Old Child Presenting with Hypercholesterolemia

Gene Express

16

DNA

SunitaR.Setlur Charles Lee

“Tumor Archaeology Reveals that Mutations Love Company”

Cell

Broad

Nature

片

段纯化、

PCR

Agilent 2100Agilent 2100

Ailent 2100

RNA QC

Agilent

2100

Agilent 2100

Agilent 2100

FDA 21CFR Part11

（Lab-on-Chip）

“Lab-on-Chip”

“Lab-on-Chip”

Caliper

Agilent

Caliper

LabChip

LabChip LabChip

X/GX II

Lab-on-Chip

Agilent 2100 RNA

RNA

RNA

G-

Gene Express

17

Agilent 2100分析系统

1% AgaroseAgilent 2100 A RNA

RNA RNAAgilent 2100

A

Agilent 2100 DNA

Agilent 2100 DNA PCR

PCR

PCR

Agilent 2100 DNA

DNA

DNA

PCR

PCR SNP

DNA

PCR GMO

PCR PCR-RFLP

RT-PCR、 Northern cDNA

Agilent 2100

RNA

(RNA Integrity Number) RNA

RIN RNA RNA

RIN RNA

RNA

PCR(qPCR) RNA

RNA

RNA (smallRNA)，

miRNAs,siRNAs, snRNAs miRNA

RNA siRNA

21nt

mRNA ’UTR

RNA

Agilent 2100 RNA

RNA

MirVana miRNA Isolation Kit

RNA A RNA 28s、

18s RNA 5.8s、5s、tRNA

miRNA、siRNAs、snRNAs)

RNA

RNA RNA

Agilent 2100

RNA 2A

A Agilent 2100 A

2B Agilent 2100

RNA miRNA

qPCR

Angiogenesis

Samples: 1 2 A 3 4 L 1 2 A 3 4

28S

18S

*

A

B

Gene Express

18

METH-2N= T=ABPPBP-2 METH-2

METH-2 ABPPBP-2

Agilent 2100

NGS

NGS NGS

DNA

DNA

Agilent

DNA DNA

DNA

DNA Agilent 2100

NGS

PCR DNA

PCR

Agilent 2100 DNA

NGS DNA QC

PCR DNA

Agilent 2100 DNA

4A Agilent 2100

PCR DNA B

PCR cycles

500bp PCR

PCR DNA Agilent 2100 PCR cycles PCR

DNA Agilent 2100 PCRcycles

pg/µL DNA

PCR

Agilent

2100

NGS DNA RNA QC

NGS QC

LabChip GX/GX II

Lab Chip GX/GX II

28s/

FDA 21CFR Part

GLP、GCP、GMP 4Q(DQ/IQ/OQ/PQ)

LabChip GX

DNA QC

Patient No 308 314 323 151 358 353Tissue N/T N/T N/T N/T N/T N/T

ABPPBP-2,control gene

METH-2,downregulatedin tumour

ladder

200 bp

150 bp

100 bp

tumourtissueMETH-2

ABPPBP-2control geneproductnormal

tissueMETH-2

[FU]

60

50

40

30

20

10

055 60 65 70 75

A

B

细血管后静脉发展而形成新的血管。血管形成是促血管形

成因子和抑制因子协调作用的复杂过程，正常情况下二者

处于平衡状态，一旦此平衡打破就会激活血管系统，在肿

瘤的发展转移过程中起到重要作用。METH-2就是其中一

种重要的抗血管形成因子，过去的芯片实验表明METH-2

在大部分非小细胞肺癌样本中表达受到抑制，研究者通过

Agilent

ETH-2多重PCR产物的表达情况（图3），结果发现与癌

旁组织相比，METH-2在23个非小细胞肺癌样品中的表达

量明显下调，验证了过去芯片实验的结果，而进一步研究

发现METH-2的表达下调与该基因启动子区甲基化有关。

2100来比较非小细胞肺癌和相应的癌旁组织中M-

Gene Express

19

LC GX DNA QC

clusters

LabChip GX qPCR

DNA

400bp+40bp

Caliper LapChip

XT 7A Caliper

Lab-on-chip

LabChip XT

7B

5)。HT DNA High Sensitivity Kit pg/µL

DNA NGS

PCR DNA

PCR

LC GX QC

LC GX

LC GX DNA RNA QC

RIN RNA LC GX

RNA (RNA Quality Score，RQS

RNA

LC GX RNA 28S、18S、

28S/18S( 28S/18S 28S/ 18S/

Fast Area Ratio RQS RIN

LC

GX RQS

LabChip GX RNA NGS RNA

LabChip XT DNA

DNA

DNA EB

DNA

LabChip XT

0.5ng DNA

LabChip XT

(skip extraction)

LC GX RQS RIN

LC XT LC XT

Gene Express

20

LCXT LCGX

（adapter dimers LabChip XT

50bp-750bp 1000bp 25bp-300bp

500bp DNA LC XT

DNA

read GC tolerance

，Caliper

XT DNA 300 Assay

adapter dimers，

小RNA库

MicroRNAs(miRNA) RNA

miRNA miRNA

RNA

LabChip XT

RNA

adapter dimers

LC XT分析小RNA的

LabChip XT LabChip

GX DNA high sense assay total input library the XT

fractionated library LC XT

adapter dimers

LabChip XT RNA

LC XT DNA 300 Assay Illumina RNA

(NEBNext 和TruSeq™)

adapter dimers Broad Ins-

titute NGS

Illumina ABI

Roche GC

Illumina Caliper Genome

Analyzer HiSeq 2000

®

Gene Express

21

Zephyr Sciclone

Zephyr Sciclone NGS

Zephyr® Molecular Biology Workstation Caliper

Caliper

PCR/

premix

Excel表格 自动计算出

(Lab Automation Workstation)

(Liquid Handler)

Zymark

Zymark Caliper

Caliper Life Sciences

Caliper

Zephyr

Sciclone

TwisterⅡ

Staccato

DNA

(NGS)

DNA

Caliper Zephyr

Sciclone NGS

Gene Express

22

Promega Wizard SV 96 Genomic DNA Purification System Zephyr

Promega Wizard SV 96 Genomic DNA Purification SystemZephyr DNA Nanodrop

PCR LabChip GX和HT DNA 5K

如何

NGS Z-

ephyr Promega、Millipore

HEPA

DNA/RNA

Zephyr

Promega Wizard SV 96 Genomic DNA Purification System

Zephyr工作台面排布

DNA Hela cells

µL

ng/µL OD

DNA

Hela cells

DNA

PCR µL ß-actin PCR TE

µL，LabChip GX

Zephyr NGS Post-PCR

Workstation

1-200µL:1-5µL CV<5%，5-200µL CV<2%

0.5-25µL:0.5-2µL CV<8% 2-25µL时CV<5%

Zephyr

Caliper pre-PCR post-PCR

Zephyr NGS Post-PCR

pre-PCR post-PCR

Zephyr

(NGS)

post-PCR qPCR

Gene Express

23

NGS

Caliper

NGS

Library Prep

µL Cold RNA

µL Caliper

Sciclone NGS WorkStation

Caliper Sciclone NGS

WorkStationCaliper NGS

Sciclone NGS WorkStation

equencing） RNA (RNA sequencing)

(ChIP‐Sequencing)

AGBT

Capture

Capture

NGS

Agilent SureSelect Capture

Library preps capture NimbleGen

Library preps capture

Whole exome sequencing Targeted res-

Sciclone Workstation

Illumina、Life Tech、Roche

Pacific

xome-Seq、Targeted-Seq

Illumina TruSeqDNA、RNA、Exome

enrichment、Nimblegen SeqCap、Agilent SureSelect

NuGen kit

Bio DNA-Seq、RNA-Seq、E-

Gene Express

24

mL µL

Labturbo

Labturbo

TAIGEN

Labturbo

Labturbo

Labturbo

PCR Labturbo

Labturbo

µg

CPU

Labturbo

qPCR Labturbo

DNA RNA

Labturbo

Labturbo

Gene Express

25

μL

PCR

Sample Yeilds (ug)

Concetra�on (ng/μl)

Whole Blood 220 μL 1000μL

3-8 15-35

15-40 75-175

Buffy Coat 200 μL 500 μL

20-40 50-120

100-200 250-600

Chicken Liver 25 mg 20-40 100-200

Hela Cell 1 x 106 15-25 75-125

Arabidopsis leaves 100 mg 2-3 10-15

Elu�on volume 200μL

PCR

Labturbo

Ct

PCR

PCR

DNA、RNA

PCR

Labturbo

μL

DNA

μL

qPCR Ct

μL

DNA

qPCR

HBV DNA Ct

Ct CV

26

qPCR CtQPCR

μL PCR

DNA，PCR

QPCR

Labturbo

qPCR

Gene Express

Gene Express

27

SMRT cell

SNP

SNP PacBio RS

PCR

PCR

GC A+T CG

PacBio RS

PacBio RS

PacBio RS

PacBio RS

1 de Novo

bp PCR

GC AT PCR

PacBio RS DNA

PCR DNA

bp GC AT

Chen-Shan Chin PacBio RS

E

DNA

Pacific Biosciences

PacBio RS，

DNA

PacBio RS SMRT Cell

(zero-mode

waveguides，ZMWs) ZMW DNA

DNA

ZMW

DNA DNA

DNA

PacBio RS系统的一级信号处理器

PacBio RS

Gene Express

2

Gene Express

28

E.coli O104:H4

NCBI

NGS

JGI

Broad

Wellcom Trust Sanger Institute

The Cold Spring Harbor Laboratory

Expression Analysis.Inc PacBio RS

PacBio RS

(Rhodopseudomonas palustris)

DX GC

PacBio RS SMRT

suffix tree

PacBio

MO10 1992 India

M989 1993 India

M988 1993 Bangladesh

M987 1992 India

M986 1992 India

M985 1992 India

M984 1992 India

M835 1993 Bangladesh

M834 1993 Bangladesh

M833 1993 Bangladesh

M831 1993 Bangladesh

M545 1993 India

M542 1993 Bangladesh

M540 1993 India

M537 1993 India

MJ-1236 1994 Bangladesh

M828 1991 Morocco

M827 1990 Guinea

M824 1987 Algeria

M791 1991 ThailandM4 (MDC126 Bangla

desh 2008) H2 (Haiti 2

010)H1 (Ha

iti 2010)CIRS-

101 2002 B

angladesh

B33 2004 Mozam

bique

M654 1991 India

M822 1983 Vietnam

M764 1989 Thailand

M740 1985 Thailand

M723 1982 Thailand

M714 1979 Bangladesh

M652 1981 India

M646 1979 BangladeshRC9 1985 KenyaN5 (N16961 Bangladesh 1971)

N16961 1971 BangladeshM825 1988 Zaire

M797 1986 H

ong Kong

M795 1976 Bangladesh

M650 1976 India

M647 1970 Bangladesh

M820 1978 MalaysiaM815 1973 PhilippinesM811 1971 Burma

M808 1969 Vietnam

M807 1966 Vietnam

M806 1964 India

M805 1963 Cambodia

M804 1962 India

M803 1961 Hong Kong

M799 1989 Hong Kong

M686 1968 Thailand

M663 1992 Indonesia

M662 1993 Indonesia

M793 1961 Indonesia

M640 1954 EgyptM543 1938 Iraq

M66-2 1937 Indonesia

Group VI

Group V

Group IVGroup III

Gro

up II

Group I

PandemicPre

–7th

M2316 1998 Peru

M2315 1999 Brazil

M2314 1991 Peru

M830 1993 French Guiana

M829 1992 Malawi

M826 1990 Malawi

M823 1984 Algeria

M821 1982 France*

M819 1975 Germany*

M818 1975 Comoro Islands

M817 1974 Chad

M816 1974 Senegal

M814 1972 Morocco

M813 1972 Senegal

M812 1971 Chad

M810 1970

Ethiopia

M809 1970 Sie

rra Leone C6 (C6706 P

eru 1991)

RC385 Chesapeake Bay 1998VL426 UK UnknownV51 United States 1987TM11079-80 Brazil 1980

12129(1) Australia 1985

62-339 Bangladesh 20021587 Peru 1994

TMA21 Brazil 1982AM19226 Bangladesh 2001

MZO-3 Bangladesh 2001MZO-2 Bangladesh 2001

V52 Sudan 1968O395 India 19652740-80 United States 1980MAK757 Celebes Islands 1937NCTC 8457 Saudi Arabia 1910BX330286 Australia 1986

B

A

di A bi 9 0stralliiaa 19986

N5 (N16961 Bangladesh 1971)

N16961 Bangladesh 1971

C6 (C6706 Peru 1991)

RC9 Kenya 1985

MO10 India 1992

B33 Mozambique 2004

MJ-1236 Bangladesh 1994M4 (MDC126 Bangladesh 2008)

CIRS 101 Bangladesh 2002

H2 (Haiti 2010)

H1 (Haiti 2010)

sepA

B C

A

Chromosome vs. TY2482

Chromosome vs. 55989 Outbreak Strain C227-11 vs. 55989

Plasmid ESBL

Plasmid AA

Structural variationregion associated withvirulence-factor genes

Structural variationregion associated withvirulence-factor genes

Lambdalikephageelements

55989 (inner track)

C777-09 (outer track)C754-09C760-09C682-09C35-10C734-09C227-11E

C04

2_pA

A01

1E

C04

2_pA

A01

1

EC042_pAA011

EC04

2_pA

A013

EC04

2_pA

A014

EC042_pAA034

EC042_pAA034

EC04

2_pA

A034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA035

EC042_pAA035

EC042_pAA035EC

042_pAA035EC

042_pAA035

EC042_pAA050

EC042_pAA050

EC04

2_pA

A050

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA158

EC042_pAA158

EC

042_pAA

021

EC042_pAA022

EC042_pAA023

EC042_pAA023

EC042_pAA023

EC042_pAA042

EC042_pAA042

ydiE(hemP)

capU

aaiC

sigA

pic

pic

air

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

EC042_pAA011

EC042_pAA011

EC042_pAA011

EC

042_pAA

013 410

AAp

_240

CE

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC04

2_pA

A034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA035EC042_pAA035

EC042_pAA035

EC042_pAA035

EC042_pAA035

EC042_pAA035

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_

pAA05

0

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA158

EC042_pAA158

EC042_pAA158EC042_pAA158

EC042_pAA021

EC042_pAA022

EC042_pAA023

EC042_pAA023

EC042_pAA042

EC042_pAA042

EC

042_

pAA

042

EC042_pAA062

ydiE(hemP)

capU

aaiC

sigA

pic

pic

air

blaCTX-M-15

blaTEM-1

sepA

aap

aggR

aggA

aatB

aatA

EC042_pAA035

EC042_pAA023

EC042_pAA042

5.0

4.5

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

ydiE(hemP)

5.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Outbreak Strain C227-11

55989

EC04

2_pA

A011

EC04

2_pA

A011

EC042_

pAA01

1

EC04

2_pA

A013

EC04

2_pA

A014

EC04

2_pA

A021

EC

042_

pAA

022

320

AAp

_240

CE E

C042_pA

A023

EC042_pAA034

EC042_pAA034

EC04

2_pA

A034

EC04

2_pA

A034

EC042_pAA034

EC042_pAA034

EC042_pAA035

EC042_pAA035

EC042_pAA035

EC042_pAA035

EC042_pAA042

EC042_pAA050

EC042_pAA050

EC04

2_pA

A050

EC04

2_pA

A050

EC042_pAA050

EC042_pAA050

EC042_pAA158

EC042_pAA158

110

AAp

_240

CE110

AAp_240

CE

EC042_pAA011

EC

042_pAA

013EC

042_pAA014EC04

2_pA

A021

EC04

2_pA

A022

EC04

2_pA

A023

EC04

2_pA

A023

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_

pAA03

4

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC042_pAA034

EC04

2_pA

A035

EC04

2_pA

A035

EC04

2_pA

A035

EC04

2_pA

A035

EC042_

pAA03

5

EC04

2_pA

A035

EC042_pAA042

EC042_pAA042

EC042_pAA042

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_

pAA05

0

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC042_pAA050

EC04

2_pA

A062

EC042_

pAA15

8

EC04

2_pA

A158

EC04

2_pA

A158

EC04

2_pA

A158

capU

aaiC

sigA

pic

pic

air

ydiE(hemP)

capU

aaiC

sigA

pic

pic

air

E.coli

David A. Rasko

David PacBio RS

E.coli

E.coli

E.coli

E.coli

DNA

bp

NGS

GC

Gene Express

29

PacBio

PacBio RS NGSaye-aye

DNA

PacBio

JGI

PacBio RS Tb DNA

GC

Gaps in Dpseudo Assembly

Gap Size

Freq

uenc

y

0

050

010

0015

0020

00

Total of 6029 Gaps. Total Gap Length 6,675,902 (5% of the Genome)

Contig N50

Scaffold N50

Total Size

D. pseudo v2.0 53,053 bp 12.52Mb 152.7Mb

Contig N50 Scaffold N50

Total Size

Parrot 134.3 Kbp 13.05 Mbp 1.17 Gbp

Gaps in Parrot Assembly

Gap Size

Freq

uenc

y

0 5000 10000 15000 20000

050

0010

000

1500

020

000

Total of 49,376 Gaps. Total Gap Length 154,912,219 (11% of the Genome)

D. pseudo v2.0

D. pseudo Upgraded

Improvement (Magnitude)

Gap Count 6,029 1,902 4,127

(68.4%)

Gap N50 3,703 bp 22,295 bp 18,592 bp

(6x)

Total Gap Size 6.67 Mb 3.12 Mb

3.55 Mb (53.2%)

Contig N50 53,053 bp 208,529 bp 155,476 bp

(4x)

Total ContigSize

146 Mb 151 Mb 5.2 Mb (3.5%)

Scaffold N50 12.5 Mb 11.7 Mb 759 Kb (-6.4%)

Total Scaffold Size

152 Mb 154 Mb 1.68 Mb

(1%)

Parrot Original

Parrot Upgraded

Improvement (Magnitude)

Gap Count 49,376 39,937 9,439

(17.1%)

Gap N50 10,546 bp 11,024 bp 478 bp

Total Gap Size 154.9 Mb 133.5 Mb

21.4 Mb (13.8%)

Contig N50 134.3 Kb 231.3 Kb 97 Kb (1.7x)

Total ContigSize

1.17 Gb 1.2 Gb 30.72 Mb

Scaffold N50 13.0 Mb 12.9 Mb 100 Kb

Total Scaffold Size

1.33 Gb 1.33 Gb ---

Low coverage (0.5X coverage) from PacBio reads & Illumina paired end reads (38X) improve assembly

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

N25 N50 N75

bp

PacBio+Illumina

Illumina Only

Total Contigs: 3,237,204 347,237 (9.3X) Total Scaffolds: 2,564,533 273,317 (9.4X)

R

R

R

R

gaps kb。

PacBio

X k subread

gaps PacBio

gap

gap

Biotechniques

denovo gap PCR

Kb gap

PacBio

SMRT Cell PacBio

Kb gap GC

hairpin

Sanger Improving genome

assemblies by sequencing PCR products with PacBio.Xiaojing

Zhang, Karen W. Davenport, Wei Gu et al. Biotechniques Vol.

53 No. 1 2012

SMRT

PacBio

N

PacBio RS

mate-pair

PacBio

anger DX

PacBio RS

PacBio

FoxP2 egr1

Nature Biotechnology

Hybrid error correction and de novo assembly of

single-molecule sequencing reads.Sergey Koren, Michael C

Schatz, Brian P Walenz et al. 1 July 2012; doi:10.1038/nbt.2280

RS S-

Schatz M 对酵母和水稻运用PacBio技术进行测序

利用二代测序和PacBio数据进行混合拼接 结果分别将

Scaffolds数减少了4%和6%。通过二代和三代数据的混合

拼接 可以大幅提高基因组拼接效率。NGS平台所获得的

海量数据

有益的信息。

Weill Cornell Christopher Mason X

PacBio RS X NGS

PacBio

Contig

可在PacBio测序步骤得到解读 从中获得更多

Gene Express

30

PacBio DNA pulse

Pacbio DNA SMRT view

motif

DNA PacBio RS

DNAG G A G A T C A G C A G A G A A C G G C T T T C T C C A G G C T T C A A A A A T A A A C A T T T T A A A T G C C A C A G A A C G T A G A G G C T G T T C A C A G T G G T T T C A T G G T G C C

G A A G A T C A G C A G A G A A C G G C T T T C T C C A G G C T T C - A A A A T A A A C A - T T T A A A T G - C A C A G A A C G - A G A G G C T G T T C A C A G T - G T C T C A T T G T G C CG A A G A T C A G C A G A G A A C - G C - T T C T C C A - G C T T C A A A A A T A A A C A - - T T A A A T G - C A A A G A A - G T A G A G G C T G T T C A C A G T - G T T T A A T G G T G C CT G A G A T C A G C A G A G A A C G G C - T T C T - C A G G C T T C A A A A A - A A A - A A T T T - A A T G C C A C A G A A C G - A - A G G C T G - T C A C A G T G C T A T C A T G G T G - CG C A G A T C A G C A G A G A A C G G C T T T C T C C A G G C T T C A A A A A T A A A C A T T T A A A A T G C C A C A G A A C G T A G A G G C T G T T C A C A G T G G T T T C A T - G T - C CG A A G A T C A G C A G A G - A C G C C T T T C T C C A T G C T T C A A A A A - A A A C A T T T T A A A T G C C A C A G - A C T T A G A - G C - G T T C A C A G T G G T T T C A T G G T G C C

T G A G A T C A G C A G A G A A C G T C - T T C - C C A G G C T T C A A A A A T A A A C - - T T T A A A T G C C A C A G A A C G T A G A G G C T G - T C A C A G T G G T T T C A T G G T G - CG A A G A T C - G C A G A G A A C - G C T T T C T C C A - G C T A C - A A A A T A A A C A T T T T A A A T G C C A C A G A A C G T A G A G G C T G T T C A C A - T G G T T T C A T G G T G G C- G A G A T C A G C A G - G A A C G G C T T T C T - C A - G C - T C - A A A A T A A A C - T T T T - A A T G C C A C A G A A - - T A G A G G C T G - T C A C A G T G G T T T C T T G G T G C C- G A A A T C A G C A G A G A A C - G C T T T C T - C A - G C T T C - A A A A T A A A A A T T T T - A A T G C C A C A G A G C G T A G A G G C T G - T C A C A G T - G T T T C A T T G T G - CG A A G A T C A G C A G G G A A C G G C T T T C T C C A - G C T T C - A A A A T A A A C - T T T T A A A T G C C A C A G A A C G T A G A G G C T G T T C A C A G T G G T T T C - T G G T G C C- G A G A T C A G C A G A G A A C - G C T T T C - C C A G G C T T C A A A A A T A A A C A - T T T A A A T G C C A C A G A C C G T A G A G G C T G T T C A C A T T G G T T T C A T G G T G C C

- G A G A T C A G C A G A G A A C G G C T T T C T C C A G G C - T C A A A A A T A A A C A - - T T A A A T G C C A C A G A A C G T A G A G G C T G - T C A C A G T - G T T T C A T G G T G C C- G A G A - C A G C A G A G A A C G G C T T T C T C C A G G C T T C - - - A A - A C A C A - - - T A A A T G - C A C A G A A C T T A G A - G C T G T T C A - A G T - G T T T C A T - G T G C C

G A A G - T C - G C A - A G - A C G G C T T T C T C C A - G C T T C - - A A A T A A A C A T T T T A A A T G - C A C A G A A C A T A G A G G C T G T T C A C A G T G G T T T C A G G G T G - C

2.50

1.25

0.00IPD (s)

45826070 45826080 45826090 45826099 45826108 45826118 45826128 45826137 45826146 45826156

Chromosome 1 position (bp)

Reference

Forward

strand

reads

Reverse

strand

reads

c

45826000 45826050 45826100 458261500

500

1,000

1,500

2,000

Time (s)

Chromosome 1 position (bp)

0 100 200 300 400 500 600 70001020304050 T

GCA

Fluorescence

intensity (a.u.)

Time (s)

700 800 900 1,000 1,100 1,200 1,300 1,40001020304050

Fluorescence

intensity (a.u.)

Time (s)

1,400 1,500 1,600 1,700 1,800 1,900 2,000 2,10001020304050

Fluorescence

intensity (a.u.)

Time (s)

R

R

R

R

R

R

F

F

F

F

F

a b Figure 3 | Example of 5-hmC detection by SMRT sequencing from mESC genomic DNA. ( a ) The raw SMRT sequencing read. A.u., arbitrary units. (b) Sequencing subreads from the SMRTbell template, mapped onto mouse chromosome 1 over the sequencing time course. Pauses appear as discontinuities as the polymerase temporarily stops progressing along the DNA template. F, forward strand reads; R, reverse strand reads. (c) Subreads are grouped and annotated by IPD values in a heat-map scale, identifying a hemi-hydroxymethylated CG position in this DNA molecule. Arrows indicate consistent pausing of the polymerase (that is, large IPD value) at the same genomic position across multiple intramolecular subreads, indicating the presence of a 5-hmC adduct.

Novel sequence motif: CTGCAG

Lambda-like phage element specific to outbreak strainContains stxABContains putative methyltransferase and restriction enzyme for CTGCAG motif

55989 (inner track)C227-11 (Outbreak strain)C734-09C35-10C682-09C760-09C754-09 C777-09 (outer track)

Novel sequence motif: CTGCAG

Matt Waldor – HarvardRich Roberts – New England BiolabsEric Schadt, Gang Fang – Mt. Sinai

Oxidative Damage

DNA DNA

DNA

PacBio RS

Nature

5-mC 5-hmC

N6-A

PacBio de

Novo EAEC

BGI

PacBio New England Biolabs

CTGCAG motif

CTGCAG CTGCAG

PacBio

Gene Express

31

PacBio DNA IPD

Target Resequencing

PCR

PacBio RS cell

PacBio RS

UV Radiation

Figure 2: Example of a figure caption

noitaidaR gnizinoInoitalyklAPCR

SNP

PacBio RS

SNP Broad

PacBio

de novo SNP(

Nature Methods

PacBio

BCR-ABL

bp

PacBio RS

BHSC PacBio

Kosrae

Kosraen

Kosraen

kb PCR扩增子 PacBio

bp bp

SMRT

G

T3 AML

PacBio FLT3

Kb

FLT3

FLT3

PacBio

Nature FL-

RS

RS

SNP

Gene Express

32

FLT3

UC Davis CGG Trace

ChIP Sequencing

DNA

DNA

PacBio RS

ChIP DNA

DNA

GC AT

GC

UC Davis

X FMR1 CGG

X

X

X FRM1

CGG

CGG X

FXTAS CGG

X FRM1

FMRP Oostra, B.A. and R. Willemsen, FMR1: a gene

with threefaces. BiochimBiophysActa,2009. 1790 (6): p. 467-77.

CGG Sanger

PacBio

CGG

PolyA A

PacBio RS PCR

ral-pallidoluysian atrophy，DRPLA)

ATN1 atrophin1 CAG

CAG

CAG

CAG

CAG

PCR CAG

GC PCR

Pacbio RS

DNA

PCR

DNA DNA

nt

DNA DNA

PacBio RS CCS SMRT cell

CAG CCS

Pre-Treatment Relapse Normal Control #1

Subject Number Mutation Native

CodonAlternative Codon

Observed Alternative Codon

Frequency in ITD+

Sequences

Total Number of ITD+ Sequences Sampled

Observed Alternative Codon FrequencyIn ITD+ Sequences

Total Number of ITD+ Sequences Sampled

Observed Alternative Codon Frequency

Total Number ofSequences Sampled

1009-003 D835Y GAT TAT 0.21% 482 8.4% 332 0.00% 768

D835V GAT GTT 0.00% 482 3.3% 332 0.13% 768

D835F GAT TTT 0.00% 482 10.2% 332 0.00% 768

1011-006 D835Y GAT TAT 0.00% 196 41.0% 402 0.00% 768

1011-007 F691L TTT TTG 0.18% 561 6.2% 341 0.22% 450

D835Y GAT TAT 0.00% 930 3.0% 436 0.00% 768

D835V GAT GTT 0.43% 930 29.6% 436 0.13% 768

1005-004 F691L TTT TTG 0.00% 496 29.6% 513 0.22% 450

1005-006 D835Y GAT TAT 0.00% 171 39.5% 261 0.00% 768

D835F GAT TTT 0.00% 171 2.7% 261 0.00% 768

1005-007 D835Y GAT TAT 0.00% 57 4.0% 378 0.00% 768

D835V GAT GTT 0.00% 57 47.4% 378 0.13% 768

1005-009 D835Y GAT TAT 0.00% 19 50.6% 445 0.00% 768

1005-010 F691L TTT TTG 0.00% 387 25.3% 150 0.22% 450

CGG repeat core

(dentatorub-

Gene Express

33

PacBio cDNA

SMRT PacBio

RNA

SMRT ZMW DNA

RNA DNA

DNA SMRT bell

SNP kb以

上 DNA

DNA CNV

FISH

PCR、Sanger

PacBio

PacBio

kb Mb

缺失位点

MDA PacBio RS

PCR SNP

PacBio RS

SNP

PacBio

RNA

cDNA

PacBio RS

[1]Chen-Shan Chin et al. The Origin of the Haitian Cholera Outbreak Strain，December 9, 2010. The New England Journal of Medicine.[2] David A. Rasko, Ph.D.et.al . Origins of the E. coli Strain Causing an Outbreak of Hemolytic–Uremic Syndrome inGermany. N Engl J Med 2011[3] Adam English et al. Mind The Gap: Upgrading Reference Genomes with Pacific Biosciences RS Long-Read Sequencing Technology. Poster[4]http://www.ebiotrade.com/speech/down/Gene120420/play2.htm[5]Schatz M. et al. Combining short (Illumina) and long (PacBio) NGS reads to improve de novo genome assemblies. Poster[6] Benjamin A. Flusberg et.al Direct detection of DNA methylation during single-molecule, realtime sequencing. Nat Methods. 2010 June ; 7(6): 461–465. [7]Chun-Xiao Song et.al. Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nature Methods, 20 November 2011.[8]Schadtetal. Modification Analysis of German E.Coli /2012_03/presenta tions.php[9]Tyson A. Clark et.al. Direct Sequencing and Identification of Damaged DNA Bases. Genome Integrity 2011, 2:10 [10]Michael Brown, Jason Chin. “BCR-ABL Haplotype Rare Variant Project.” Menlo Park, CA: Pacific Biosciences, 2010.[11]Adam English et.al. SMRT Sequencing of Whole Mitochondrial Genomes and Its Utility in Association Studies of Metabolic Disease.Poster[12]Catherine C. Smith et. al. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature[13]Paul Hagerman MD. Sequencing the Unsequenceable: Expanded CGG Repeats in the Human FMR1com/forms/webinar_paul_hagerman.html[14]Jason Walker et.al. 1Single-Molecule, Real-Time (SMRT®) DNA sequencing facilitates long trinucleotide repeat characterization.

eakStrain Yields New Insights. http://www.pacb.com/newsletter

Gene. http://www.pacificbiosciences.

Outbr-

Gene Express

34

Laser Micro Dissection LMD

DNA、RNA

MMI Molecular Machines & Industries

(CellCut Plus SmartCut Plus)

(CellEctor Plus) (Cell Manip-

ulator Plus) MMI

MMI CellCut Plus

nm

X µm

XY

µm

Serial Section Function

RNA

CellCut Plus

PTP,

Positive Target Positioning

Serial Section

Gene Express

35

Z-Drill

Z-Drill Function

CellCut Plus X/Y

Z Z

Z-Drill

CellEctor Plus MMI

CCD XY

CCD

CellEctor Plus

nL

MMI PCR

Realtime PCR CellEctor Plus

DNA-free Ampligrid

Amplispeed SlideCycler Preamplification

Realtime PCR DNA

CellManipulator Plus

Schipper H HMM

nf bp

bp

nf bp

NF HMM

(Schipper H et al,Mutational analysis ofthe nf2 tumour

suppressor gene in three subtypes of primary humanmalignant

Cell Manipulator Plus

pulator Plus optical tweezer

nm W

PN

μm μm Z

MBPS-Multibead positioning detector

QD quadrant detector

DNA

Actin/myosin

pipettingmaster mix

Loading 1μlto AmpliGriddemo slide

Covering mastermix with 5μl

sealing solution

transferring slideto

themal cycler

PCRamplification

addingloading dyeto reaction

Rummingagarose gel

Result ofchr 21

specific PCR

CellMani-

Gene Express

36

Pugh,T.J et al,Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefitinib response in a retrospective analysis of lung cancer patients.2007 Cancer7,128

Sánchez-Aguilera A et al,Silencing of the p18INK4c gene by promoter hypermethylation in Reed-Sternberg cells in Hodgkin lymphomas.2004,Blood 103,2351-2357

mesotheliomas.2003,Int.J.Oncol.22,1009-1017）

Gefitinib

FR Pugh

TJ EGFR、HER2 Gefitinib

gefitinib

DNA EFGR

FISH EGFR HER2

EGFR HER2 EGFR、

HER2 Gefitinib

palmar fidromatosis Dupuytren

Wang L

MMI

DNA X

PCR X

(ZhuHetal, Clonal analysis of palmar fibromatosis: a study

whether palmarfibromatosis is a real tumor. J.Transl. Med

2006,4,21）

4 p18INK4c CDK CDK6/CDK4

p18INK4c B

p18INK4c

Sánchez-Aguilera p18INK4c

p18INK4c

Reed-Sternberg RS

B p18INK4c

p18INK4c p18INK4c RS

RS DNA

PCR p18INK4c

p18INK4c

p18INK4c

p18INK4c

RS

LMD

MMI CellCut Plus

，CellEctor Plus Cell Manipulator

Plus

BA C

EG-

37

Gene Express

Covaris

Covaris CryoPrep

Covaris AFA

Covaris AFA

Covaris AFA

Covaris

CryoPrep

CryoPrep

CryoPrep

AFACovaris AFA Covaris

AFA

DNA Covaris AFA

M、S、E、L

Covaris AFA

CryoPrepCryoPrep

CryoPrep

CryoPrep

CryoPrep

Gene Express

38

Covaris AFA

Covaris

Covaris

CryoPrep

CryoPrep

RNA RNA

CryoPrep

Covaris RNA

DNA/RNA

RNA Covaris AFA

RNA

Covaris

Covaris AFA

Covaris

Covaris

Gene Express

39

TM

Eco PCRCq

Average

Cq

Standard

Devia�on of Cq

Maximum

Cq

Minimum

Cq

24.131 0.063 24.339 24.017

Eco PCR

LED

LED

LED

CCDTMEco PCR

PCR

CV

PCR PCR

Cq

Eco

Eco PCR

(nm) (nm)

1 452 - 486 505 - 545 SYBR Green I, FAM

2 452 - 486 562 - 596 HEX, JOE, VIC

3 542 - 582 604 - 644 ROX, Texas Red

4 542 - 582 665 - 705 Cy5, Quasar 670

Eco PCRTM

Eco PCRTM

EcoTM

Eco PCR Illumina

PCR

David Baltimore

Axel Scherer

Eco PCR

PCR

High Resolution Melt(HRM)

Eco PCR Netbook

TM

Gene Express

40

Eco PCRHRM

Eco PCR

Eco PCR

Eco PCRSYBR

EcoExcel,

CSV, PDF PPT ； .jpeg .bmp

Eco

MIQE（The Minimum Informationfor Publication of Quantitative

Real-Time PCR Experiments） PCR

Eco PCR PX2

∆∆

Eco PCR

Real-Time PCR

Cq

（HRM）

Eco

PCR R Eco

PX2 log

Slope PCR

R

Eco PCR

Forman O.P Illumina

Eco PCR FAM83H BCAN

ACTB

BCAN

FAM83H

∆∆ Cq

HRM

Eco Real-Time PCR

System High Reso-

Gene Express

41

FAM83H BCANACTB

HRMM.carinatus

M. carinatus( M holotrachys M. whitsoni D：M. spp

TM

PCR仪 PCR DNA

PCR

ECOTM Real-Time

1.Forman,O. P.,Penderis, J., Hartley, C., Hayward, L. J., Ricketts, S.L.

& Mellersh, C. S. (2012) Parallel Mapping and Simultaneous

Sequencing Reveals Deletions in BCAN and FAM83H Associated with

Discrete Inherited Disorders in a Domestic Dog Breed. PLoS Genet, 8,

e1002462.

2.TaylorFC.Mutation scanning using high-resolution melting.

Biochem Soc Trans, 2009, 37, 433-437.

3.E.M.Fitzcharle. Rapid discrimination between four Antarctic fish

species, genus Macrourus, using HRM analysis. FisheriesResearch .

2012.02.002

lution Melt (HRM)

HRM

PCR LC Green

plus PCR DNA

DNA

DNA DNA

DNA GC GC

Tm

Eco PCR HRM

E.M.Fitzcharle

HRM

E.M.Fitzcharle Illumina

Gene Express

42

sam5

sam5

sam5

E-Cadherin Ep-CAM

sam5 BLUE

SAW Instruments GmbH sam5

Surface Acoustic Wave，SAW sam5

SAW

surface plasmon resonance SPR

SPR

sam5

sam5

sam5 green samX

samX

channels

sam5 SAW

igital Transducers IDT

pg/mm

phase

Viscoelastic

amplitude

sam5

sam5 GREEN samX sam5 BLUE

sam5 blue GREEN samX

sam5 sam5 BLUE

Interd-

2 realmass

Gene Express

43

2D-COOH SAM

DMSO

DMSO

sam5

Tom Blundell

sam5 DMSO

MB605

sam5

sam5sam5

Wang, X sam5

EMA

sam5

sam5

Christopher

PL1 CAPAN2

ng

Tom CM-dextranMB605 (5μM，1.25μM，

2.5μM)DMSO MB605

Gene Express

44

sam5

[1] Wang,X.,et al. Conformational chemistry of

surface-attachedcalmodulin detected by acoustic shear wave

propagation, Mol. Bio. Syst. 2: 184-192 (2006)

[2]Christopher D. CorsoBSa, Desmond D. Stubbs

MSb,.Real-time detection of mesothelin in pancreatic cancer

cell linesupernatant using an acoustic waveimmunosensor.

Cancer Detection and Prevention; 30: 180–187 2006 .

truChIP™

CryoPrepAFA-Series

The Covaris ChIP Process--From Tissue/Cultured Cell to Sheared Chromatin

Snap freeze fresh tissue sample

Dry pulverize sample into a fine powder

Prepare sample for extraction with Covaris truCHIP Kit

Shear chromatin with highly controlled AFA acoustic energy Obtain reproducible shearing results that your assay can rely on

CytoScan HD ArrayCytoScan 750K Array

Kitted Reagents

(ChASChromosome Analysis Suite

) Software Instrumentation

GCS 3000

GCS 3000 Dx2

SNP

y aan HHDD ArrayyCyytooSScaann 77500K ArrayyCCyttooSc

SNP

Instrumenntaatioon

GCS 300000

GCS 300000 Dxx22

Kitteed Reaaggeenttss