上海市臨檢中心 PCR培訓(xùn)課件 8. illumina-NGS測(cè)序原理及質(zhì)量控制

Illumina

NGS測(cè)序原理&質(zhì)量控制Shibo

Wang(王詩博),Ph.D.Clinicalsales

Specialistillumina2018?2013Illumina,

Inc.

Allrights

reserved.Illumina,IlluminaDx,

BaseSpace,BeadArray,

BeadXpress,

cBot,CSPro,DASL,DesignStudio,

Eco,GAIIx,Genetic

Energy,Genome

Analyzer,GenomeStudio,GoldenGate,

HiScan,HiSeq,

Infinium,iSelect,

MiSeq,Nextera,NuPCR,

SeqMonitor,

Solexa,

TruSeq,

TruSight,

VeraCode,the

pumpkin

orange

color,

andthe

Genetic

Energystreamingbasesdesign

aretrademarksorregistered

trademarksof

Illumina,

Inc.

Allotherbrandsandnames

contained

hereinare

the

property

oftheir

respective

owners.illumina

測(cè)序?qū)嶒?yàn)流程illumina二代測(cè)序儀生物學(xué)意義BiologicalMeaning簇生成Clustering測(cè)序Sequencing數(shù)據(jù)分析Analysis測(cè)序樣品制備2測(cè)序樣品制備是測(cè)序成功的關(guān)鍵！樣本制備：在需要測(cè)序的DNA兩端加上與測(cè)序儀配合的接頭Dual

Index

Library

shown

控制全部DNA片段在一定范圍內(nèi)，比如：300-400bp

在DNA片段兩端加上已知序列的illumina接頭序列

加接頭的過程同時(shí)也把不同樣品DNA進(jìn)行index標(biāo)簽標(biāo)記Which

kitto

useforNGSin

different

applications?測(cè)序樣品制備SamplePrepNGS測(cè)序應(yīng)用?

客戶的研究目的？?

樣品本身的特點(diǎn)？?

可以承受的成本？4無法確定使用何種試劑盒？可以使用在線選擇工具“Illumina

SamplePrepKitSelectorTool”5Illumina

SequencingWorkflowCluster

GenerationcBotMiSeq6Whatis

a

Flow

Cell?流動(dòng)槽(Flow

Cell)是什么?A

flowcell

is

athick

glass

slidewith

channelsorlanes一種含有通道的厚玻璃片Clustergeneration

occurson

aflowcell簇生成在流動(dòng)槽（flow

ce

）上完成Eachlane

is

randomly

coatedwithalawn

o

oligos

that

arecomplementary

tolibraryadapters每條通道中都隨機(jī)植入了能與文庫接頭互補(bǔ)結(jié)合的大量短DN

片段7InstrumentationSingleDNAAmplifiedClonalLibraryClustercBotSequencer8Hybridize

Fragment&ExtendAdaptersequenceSingle

DNA

librariesarehybridized

toprimerlawnBoundlibrariesthenextendedby

polymerasesSurface

offlowcellcoated

with

alawnofoligo

pairs3’extension9Denature

Double-strandedDNANewlysynthesizedstrandOriginaltemplateDoubl

strandedmolecule

is

denaturedOriginaltemplate

washedawaydiscardNewlysynthesized

strandis

covalently

attachedtoflowcell

surface10Hybridize

Fragment&ExtendNOTE:Single

moleculesbindtoflowcell

inarandom

pattern11BridgeAmplificationSingl

strandedmolecule

flips

overandforms

abridgeby

hybridizingtoadjacent,complementary

primerHybridized

primerisextended

by

polymerases12BridgeAmplificationDoubl

strandedbridgeisformed13Denature

Double-strandedBridgeDoubl

strandedbridgeisdenaturedResult:Two

copiesof

covalently

boundsingl

strandedtemplates14BridgeAmplificationSingl

strandedmolecules

flipovertohybridize

toadjacent

primersHybridized

primerisextendedby

polymerase15BridgeAmplificationBridgeamplification

cyclerepeated

untilmultiplebridgesareformed16LinearizationdsDNA

bridgesaredenatured17Reverse

StrandCleavageReversestrands

cleaved

andwashed

away,

leaving

aclusterwith

forward

strands

only18BlockingFree

3’

endsareblocked

toprevent

unwanted

DNApriming19Read

1

PrimerHybridizationSequencing

primerishybridizedtoadapter

sequenceSequencingprimer20Illumina

SequencingWorkflowHiSeqHiScan

SQGA

IIxSequencingMiSeq21SequencingBySynthesisAdd

4F

NTP’s+PolymeraseIncorporatedF-NTP

imagedTerminator

&fluorescentdye

cleavedfrom

F

NTPX36

25122Clusters100

Microns23Illumina

SequencingTechnology

Overview3’

5’DNA(0.1-5.0

μg)ATGTACGATCGTGCAGTCACCCGATAGCSingle

molecule

array5’Library

PreparationCluster

GrowthSequencing123456789TG

TACG

AT…BaseCallingImage

AcquisitionPairedEnd

Sequencing25PairedEndSequencingSequencedstrandBlocked3’-endsSequenced

strand

isstrippedoff3

endsof

templatestrands

andlawn

primersareunblocked26PairedEndSequencingSingl

strandedtemplateloopsovertoform

abridgebyhybridizingwithalawn

primerBridgeformation3

endsof

lawnprimer

isextended3’

extension27PairedEndSequencingDoublestrandedDNA28PairedEndSequencingBridgesarelinearized

andthe

originalforward

templateis

cleavedOriginalforwardstrand29PairedEndSequencingBlocked3’-endsFree

3’

endsof

thereversetemplate

andlawnprimers

areblockedtoprevent

unwanted

DNAprimingSequencingprimerSequencing

primerishybridized

toadaptersequenceReversestrandtemplate30SequencingBySynthesis

2ndReadAdd

4F

NTP’s+PolymeraseIncorporatedF-NTP

imagedTerminator

&fluorescentdye

cleavedfrom

F

NTPX36

25131SamplePrepisCritical

forSuccessfulsequencingDualIndex

Library

shownThe

aimofthesampleprep

stepisto

obtainnucleicacid

fragmentswith

adapters

attachedonboth

ends32Sequencing

PairedEnd

Libraries

with

SingleIndexRead33SequencingPaired

EndLibraries

with

Single

Index

Read31Paired

EndTurnaround2Single

Index

Sequencing

Utilizes

3Sequencing

Reads34Illumina

SequencingWorkflowICS/RTACAS

AVAMSRData

AnalysisBaseSpace38PrimaryandSecondary

Analysis

OverviewAnalysisTypeSequencingOutputsSoftwareICS/RTAImages/TIFF

filesPrimary

AnalysisIntensities

Base

CallingICS/RTASecondary

AnalysisAlignments

andVariant

Detection39文庫質(zhì)控概述40Illumina測(cè)序流程樣本制備文庫質(zhì)控cBotMiSeqNextSeqHiSeq

2500簇生成測(cè)序HiSeqHiScan

SQGA

IIxMiSeqNextSeq數(shù)據(jù)分析41Critical

forHighQuality

SequencingData42Library

Quality

Control

andQuantificationQuality

QuantityqPCRGelQubitBioanalyzerBioanalyzer43利用Bioanalyzer檢查文庫質(zhì)量（Quality）Bioanalyzer

2100

Assay

OptionsDNA

1000ChipHighSensitivity

DNA

Chip檢測(cè)范圍25-1000

bp50-7000

bp5-500

pg/μl使用1微升稀釋后的文庫(根據(jù)文庫產(chǎn)量稀釋：10X~50X)0.1–50ng/μl使用未稀釋1微升文庫檢測(cè)樣本量44標(biāo)準(zhǔn)的Bioanalyzer

Trace結(jié)果Upper

MarkerLower

MarkerSample

PeakBaseline基線45測(cè)序文庫的定量方法無選擇性地檢測(cè)所有的核酸污染物會(huì)使結(jié)果數(shù)值升高UV-

spectrophotometerNanodrop?

僅在文庫非常集中時(shí)使用?

使用DNA

1000

chip時(shí)比較精確Bioanalyzer

2100Fluorescence-basedds-DNA

assayQub