上海市臨檢中心 PCR培訓(xùn)課件 7、Thermo張紅-半導(dǎo)體高通量測序技術(shù)及質(zhì)量控制

半導(dǎo)體高通量測序技術(shù)及質(zhì)量控制張紅賽默飛世爾科技（中國）The

world

leader

inserving

scienceFor

Research

UseOnly.Notforuse

in

diagnostic

procedures.測序技術(shù)的發(fā)展First

Gen.Sequencing

PlatformNext

Gen.

Sequencing

Platform3rd/4th

Gen.

Sequencing

PlatformANDMORE…

…2基于半導(dǎo)體技術(shù)的高通量測序原理直接檢測氫離子，并將其轉(zhuǎn)換為可由電晶體基片讀取出來的電信號(hào)。dNTPH+?

pH?

QSensingLayerSensorPlate?

VBulkDrain

SourceSilicon

SubstrateTo

columnreceiverRothberg

J.M.

etal

Naturedoi:10.1038/nature102423半導(dǎo)體高通量測序：實(shí)驗(yàn)流程簡單、快速，嚴(yán)格的質(zhì)控樣本收集至突變報(bào)告，整體檢測流程，僅需三個(gè)工作日取樣抽提文庫構(gòu)建高通量測序數(shù)據(jù)分析注釋OncomineKnowledgebaseMagMAXTM試劑盒AssayIon

TorrentIon

Reporter?

可提取cfTNA,RNA,

DNA?

高回收率?1-20

ng?

數(shù)據(jù)精確?

通量靈活?

快速測序?

檢測低至0.1%的低頻突變微量起始，僅需?

市面最大的腫瘤基因組信息數(shù)據(jù)庫，注釋突變信息，用藥指導(dǎo)組織樣本血液樣本?

多位點(diǎn)檢測(>100)QCMetricsQubitand

EgelQubitBlood抽血管、離心需求等QPCR推薦測序通量FFPE樣本的腫瘤比例、切片厚度、切片數(shù)量等芯片類型Ion318Ion520Total

Reads≥4,000,000≥4,000,000≥15,000,000QPCREqualizer

kitBioanalyzerBioanalyzerIon53051.樣本制備及質(zhì)量控制樣本制備核酸抽提文庫構(gòu)建模板制備測序數(shù)據(jù)分析Y/N）樣本類型腫瘤百分比及范圍需要的片子數(shù)目是否需要染色及微切割（≥50%20%≤?<50%10%≤?<20%<10%1-2≥2-5≥6-10重新提供片子NY大標(biāo)本Y重新提供片子≥50%20%≤?<50%<20%≥6≥10NN小標(biāo)本或穿刺樣本(22-25

Gneedle)make

new

slidesmake

new

slides樣品類型檢測內(nèi)容(assay)切片厚度腫瘤比例是否微切割使用切片數(shù)量核酸產(chǎn)量癌種固定液石蠟包埋大組織肺腺癌OFA

panel10%NBF3um70%否71ug切片結(jié)腸癌肺腺癌OST

panelOFA

panel10%NBF10%NBF5um5um83%否否3320ng105ng穿刺組織>90%1062.核酸提取樣本制備核酸抽提文庫制備模板制備測序數(shù)據(jù)分析FFPESlide

orCurlDeparaffinization

and

PKdigestionAddethanolLysisSave

flow-throughDNARNAStep

4:RecoverRNA

from

flow-throughStep

5:RecoverDNA

from

filtercartridgeStep

1:Deparaffinize

FFPEsectionsorslidesStep

2:ProteaseDigestionStep

3:NucleicAcid

Binding72.1核酸提取的質(zhì)控之Qubit定量法82.2核酸的質(zhì)控之電泳判別法DNA總量>100ng30-100ng10-30ng<10ng檢測方法電泳20ng檢測降解程度電泳10ng檢測降解程度，剩余樣本真空濃縮至8ul左右不電泳，真空濃縮至8ul左右不電泳，真空濃縮至8ul左右DNA電泳檢測降解程度評(píng)判A（合格）主條帶位置彌散帶主要分布區(qū)域大于7500bp大于7500bp600-7500bp500bp左右B（合格）C（風(fēng)險(xiǎn)）600-7500bp500bp左右D（不合格）500bp以下500bp以下（500bp以上有小部分拖尾也判D）DNA

提取總量>30ng提取合格與否結(jié)合電泳評(píng)判合格（A,B）、風(fēng)險(xiǎn)（C）、不合格（D）10-30ng<10ng未電泳，風(fēng)險(xiǎn)未電泳，不合格92.3核酸提取的質(zhì)控之Agilent

2100法103.文庫構(gòu)建樣本制備核酸抽提文庫制備模板制備測序數(shù)據(jù)分析Genomic

DNA超高多重PCR技術(shù)Ion

AmpliSeqShearDNAFragmentsLigation

&Nick-repairSizeSelect+AAOrP1P1StandardAdaptersBarcodeAdapters113.1構(gòu)建好的文庫進(jìn)行質(zhì)控質(zhì)控方法試劑盒/方法靈敏度?

gDNA

checkE-Gel?

System?5ng

/μLAmplification

checkBioanalyzer?

2100DNA

1000

chip?

DNA

HS

chip(sheared

gDNA

&sizeselected

library)0.1

-50

ng

/μL

(50

bp-7kb)10

pg/μL

-100

ng/μLDNA

HSchipQubit??Quant-iTTM

dsDNA

High

sensitivity

kits(general

inprocess

checking)FluorometerHighsensitivityBroad

range?

Ion

Library

Quantitation

Kit(Template

Dilution

factor)qPCR0.005

ng

/μL123.2構(gòu)建好的文庫質(zhì)控之Equalizer法2)

抓取3)

洗脫1)

擴(kuò)增134.模板制備樣本準(zhǔn)備核酸抽提文庫制備模板制備測序數(shù)據(jù)分析PrimersdNTPs擴(kuò)增PolymeraseMgCl21

10

0油包水PCR14Cycle1,

AnnealingCycle1,ExtensionCycle2,DenatureCycle2,

AnnealingPolymeraseTemplateCycle2,ExtensionCycle3,DenatureCycle3,

AnnealingCycle3,ExtensionCycle4,DenatureCycle

4,

AnnealingISPCycle4,ExtensionAnd

soon…PrimerOilAqueous154.測序樣本準(zhǔn)備核酸抽提文庫制備模板制備測序數(shù)據(jù)分析測序引物退火測序酶結(jié)合珠子落入孔內(nèi)上機(jī)測序16測序原理dNTP?

四種核酸分子依次流過Ion

Chip?

(半導(dǎo)體芯片)?

每個(gè)小孔均有一個(gè)傳感器記錄每次測序反應(yīng)的信號(hào)?

直接檢測自然的DNA延伸過程H+?

單張芯片每次檢測百萬計(jì)的測序反應(yīng)?pH?QSensingLayerSensor

Plate?VBulkDrainSilicon

SubstrateSourceTo

columnreceiver17T

A

C

G

T

A

C

G

T

A

C

G

T

A

C

G

T

A

C

G…etcCycle

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3TACG-----Primer------A

GTCA

A

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T

CCC

A

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Sequence

Sequence

ofInterest...---Ion

Sphere?Cycle

118T

A

C

G

T

A

C

G

T

A

C

G

T

A

C

G

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A

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C-----Primer------A

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A

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CCC

A

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Sphere?Key

Sequence

Sequence

ofInterestCycle

219T

A

C

G

T

A

C

G

T

A

C

G

T

A

C

G

T

A

C

G…etcCycle

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C

A

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A

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CCC

A

T

G...---Ion

Sphere?Key

Sequence

Sequence

ofInterestCycle

3+20T

A

C

G

T

A

C

G

T

A

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G

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A

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G

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A

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A

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Sphere?Key

Sequence

Sequence

ofInterestAndsoon…21TTTAAKey

SequenceT

C

AG22測序結(jié)果的質(zhì)控參數(shù)%Loading?

Atleast60%?

35%orless?

slightly

higher

iflow

quality

isunder

10%%PolyclonalLowQualityTotal

Reads?

Under20%forDNA

run;lower

ifhigh

polyclonal?

higher

forRNA

due

todegradedtemplate?

DNA

+RNA

=5,000,000+?

RNA

Fusions

only=2,000,000+23測序結(jié)果的質(zhì)控參數(shù)測序通量符合芯片規(guī)格要求Navigation

barBarcodingtable(if

applicable)Unaligned/Aligned

metrics推薦測序通量Chip

TypeIon314v2BCIon316v2BCIon318v2BCIon520TotalReads≥400,000Plugin

summariesOutput

files≥2,000,000≥4,000,000≥4,000,000≥15,000,000≥60,000,000Ion530Ion54024測序結(jié)果的質(zhì)控參數(shù)測序片段讀長符合該panel的amplicon實(shí)際長度?

樣品測序深度（Mean

Depth）符合實(shí)驗(yàn)設(shè)計(jì)>2000?

在靶率（On

Target）>90%

*?

擴(kuò)增子均一度（Uniformity）>90%

**FFPE樣品根據(jù)樣品DNA質(zhì)量狀態(tài)，相應(yīng)數(shù)據(jù)可能會(huì)低于新鮮組織樣品255.數(shù)據(jù)分析樣本制備核酸提取文庫制備模板制備測序數(shù)據(jù)分析TorrentServerIonReporter或其它分析平臺(tái)設(shè)置運(yùn)行模式運(yùn)行初級(jí)數(shù)據(jù)分析次級(jí)數(shù)據(jù)分析深度分析IonTorrent?PGM?

SequencerTorrent

Browserand

PluginsTorrent

ServerTorrent

ServerRunAssessmentAlignmentsQualityScoresVariantCallsDataDelivery(BAM)To

BasesTorrent

Suite?

SoftwareRunAnalysis26數(shù)據(jù)分析結(jié)果AlleleSourceChrom

PositionRefVariantAlleleCallFrequency

TypeAlleleNameGene

IDchr9

80409443chr9

80409448chr17

7577559chr17

7577558chr17

7577559chr17

7577559G-TTGA-HeterozygousHeterozygousHeterozygousHeterozygousHeterozygousHeterozygous57.150SNPINSNovelNovel------GNAQGNAQTP53TP53TP53TP53GGGG53.313.111.822SNPDELDELSNPNovel---Hot