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Illumina测序平台原理&文库构建方法介绍Shibo
Wang(王诗博),Ph.D.ClinicalSupport
SpecialistilluminaOtc6,2017©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.全球>90%的测序数据产生于
llumin
测序平台全球
70%装机市场占有率2illumina引领高通量测序行业的发展迄今最为简单经济的新一代测序首台且唯一一台高通量测序系统Miseq
Dx获美国FDA认证Mineseq问世公司成立，总部位于加州圣地亚哥市，主要业务为芯片Hiseq2000上市，高通量测序平台黄金标准HiseqX
Ten上市,宣告1000美金人基因组实现公司上市，登陆NASDAQ证券交易市场Hiseq2500登陆市场,全面加速测序进程NovaSeq6000未来将人基因组成本推向百美元Nextseq500市场，更广泛的通量范围，适合最广泛的应用收购Solexa公司，台式高通量测序系统Miseq
登陆市场首台高通量测序平台GA登陆市场1998
2000
2007201420172010
2011
2012
20132016>2,300雇员>1200名研发人员>400技术支持人适用于各种规模
&各类应用的NGS测序系统NovaSeq6000HiSeqXTen仪器价格及其产出逐渐提高HiSeq2500600
Gb
2
B|2x1501800Gb
|
6B|2x150NextSeq1000Gb
|
4B|2x125MiSeq120Gb
|400M|2x150MiniSeq15Gb
|25M|2x3007.
Gb
|25M
2
1
0单位价格降低/Gb4Sequence
what
you
want！Clinical
Research全基因组测序转录组测序全编码区RNA测序基于探针捕获的靶向测序(百kb~15Mb)TranslationalDiagnostics基于扩增子的RNA靶向测序(12~1000
targets)靶向测序(Targets
100s)PCR扩增测序SmallRNATargetsRNADNA5illumina
测序流程illumina二代测序仪生物学意义BiologicalMeaning簇生成Clustering测序Sequencing数据分析Analysis测序样品制备6测序样品制备是测序成功的关键！样本制备：在需要测序的DNA两端加上与测序仪配合的接头DualIndex
Library
shown
控制全部DNA片段在一定范围内，比如：300-400bp
在DNA片段两端加上已知序列的illumina接头序列
加接头的过程同时也把不同样品DNA进行index标签标记TruSeq
WorkflowTruSeqNano
DNAFragmentEndRepairBead-BasedSize
SelectionAdenylate3’
endsLigate
AdaptorsTruSeq
DNA
PCR-FreeEnrichDNAFragments8Nextera
Workflow9Nextera
RapidCapture
WorkflowFast,
Flexible,
Efficient10Which
kitto
useforNGSin
different
applications?测序样品制备SamplePrepNGS测序应用•
客户的研究目的？•
样品本身的特点？•
可以承受的成本？11无法确定使用何种试剂盒？可以使用在线选择工具“Illumina
SamplePrepKitSelectorTool”12Illumina
SequencingWorkflowCluster
GenerationcBotMiSeq13Whatis
a
Flow
Cell?流动槽(Flow
Cell)是什么?A
flowcell
is
athick
glass
slidewith
channelsorlanes一种含有通道的厚玻璃片Clustergeneration
occurson
aflowcell簇生成在流动槽（flow
ce
）上完成Eachlane
is
randomly
coatedwithalawn
o
oligos
that
arecomplementary
tolibraryadapters每条通道中都随机植入了能与文库接头互补结合的大量短DN
片段14InstrumentationSingleDNAAmplifiedClonalLibraryClustercBotSequencer15Hybridize
Fragment&ExtendAdaptersequenceSingle
DNA
librariesarehybridized
toprimerlawnBoundlibrariesthenextended
by
polymerasesSurface
offlowcellcoated
with
alawnofoligo
pairs3’extension16Denature
Double-strandedDNANewlysynthesizedstrandOriginaltemplateDoubl
strandedmolecule
is
denaturedOriginaltemplate
washedawaydiscardNewlysynthesized
strandis
covalently
attached
toflowcell
surface17Hybridize
Fragment&ExtendNOTE:Single
moleculesbindtoflowcell
inarandom
pattern18BridgeAmplificationSingl
strandedmolecule
flips
overandforms
abridgeby
hybridizingtoadjacent,complementary
primerHybridized
primerisextended
by
polymerases19BridgeAmplificationDoubl
stranded
bridgeisformed20Denature
Double-strandedBridgeDoubl
strandedbridgeisdenaturedResult:Two
copiesof
covalently
boundsingl
strandedtemplates21BridgeAmplificationSingl
strandedmolecules
flipovertohybridize
toadjacent
primersHybridized
primerisextended
by
polymerase22BridgeAmplificationBridgeamplification
cyclerepeated
untilmultiplebridgesareformed23LinearizationdsDNA
bridgesaredenatured24Reverse
StrandCleavageReversestrands
cleaved
andwashed
away,
leaving
aclusterwith
forward
strands
only25BlockingFree
3’
endsareblocked
toprevent
unwanted
DNApriming26Read
1
PrimerHybridizationSequencing
primerishybridizedtoadapter
sequenceSequencingprimer27Illumina
SequencingWorkflowHiSeqHiScan
SQGA
IIxSequencingMiSeq28SequencingBySynthesisAdd
4F
NTP’s+PolymeraseIncorporatedF
-NTP
imagedTerminator
&fluorescentdye
cleavedfrom
F
NTPX36
25129Clusters100
Microns30Illumina
SequencingTechnology
Overview3’
5’DNA(0.1-5.0
μg)ATGTACGATCCGTGCAGTCACCCGATAGCSingle
molecule
array5’Library
PreparationCluster
GrowthSequencing123456789TG
TACG
AT…BaseCallingImage
AcquisitionPairedEndSequencing32PairedEndSequencingSequencedstrandBlocked3’-endsSequenced
strand
isstrippedoff3
endsof
templatestrands
andlawn
primersareunblocked33PairedEndSequencingSingl
strandedtemplateloopsovertoform
abridgebyhybridizingwithalawn
primerBridgeformation3
endsof
lawnprimer
isextended3’
extension34PairedEndSequencingDoublestrandedDNA35PairedEndSequencingBridgesarelinearized
andtheoriginalforward
templateis
cleavedOriginalforwardstrand36PairedEndSequencingBlocked3’-endsFree
3’
endsof
thereverse
template
andlawnprimers
areblockedtoprevent
unwanted
DNAprimingSequencingprimerSequencing
primerishybridized
toadaptersequenceReversestrandtemplate37SequencingBySynthesis
2ndReadAdd
4F
NTP’s+PolymeraseIncorporatedF
-NTP
imagedTerminator
&fluorescentdye
cleavedfrom
F
NTPX36
25138SamplePrepisCritical
forSuccessfulsequencingDualIndex
Library
shownThe
aimofthesampleprep
stepisto
obtainnucleicacidfragmentswith
adapters
attached
onboth
ends39Sequencing
PairedEndLibrarieswith
SingleIndexRead40SequencingPaired
EndLibraries
with
Single
Index
Read31Paired
En
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