上海市临检中心 PCR培训课件 7、Thermo张红-半导体高通量测序技术及质量控制

半导体高通量测序技术及质量控制张红赛默飞世尔科技（中国）The

world

leader

inserving

scienceFor

Research

UseOnly.Notforuse

in

diagnostic

procedures.测序技术的发展First

Gen.Sequencing

PlatformNext

Gen.

Sequencing

Platform3rd/4th

Gen.

Sequencing

PlatformANDMORE…

…2基于半导体技术的高通量测序原理直接检测氢离子，并将其转换为可由电晶体基片读取出来的电信号。dNTPH+∆

pH∆

QSensingLayerSensorPlate∆

VBulkDrain

SourceSilicon

SubstrateTo

columnreceiverRothberg

J.M.

etal

Naturedoi:10.1038/nature102423半导体高通量测序：实验流程简单、快速，严格的质控样本收集至突变报告，整体检测流程，仅需三个工作日取样抽提文库构建高通量测序数据分析注释OncomineKnowledgebaseMagMAXTM试剂盒AssayIon

TorrentIon

Reporter•

可提取cfTNA,RNA,

DNA•

高回收率•1-20

ng•

数据精确•

通量灵活•

快速测序•

检测低至0.1%的低频突变微量起始，仅需•

市面最大的肿瘤基因组信息数据库，注释突变信息，用药指导组织样本血液样本•

多位点检测(>100)QCMetricsQubitand

EgelQubitBlood抽血管、离心需求等QPCR推荐测序通量FFPE样本的肿瘤比例、切片厚度、切片数量等芯片类型Ion318Ion520Total

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

kitBioanalyzerBioanalyzerIon53051.样本制备及质量控制样本制备核酸抽提文库构建模板制备测序数据分析Y/N）样本类型肿瘤百分比及范围需要的片子数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大标本Y重新提供片子≥50%20%≤?<50%<20%≥6≥10NN小标本或穿刺样本(22-25

Gneedle)make

new

slidesmake

new

slides样品类型检测内容(assay)切片厚度肿瘤比例是否微切割使用切片数量核酸产量癌种固定液石蜡包埋大组织肺腺癌OFA

panel10%NBF3um70%否71ug切片结肠癌肺腺癌OST

panelOFA

panel10%NBF10%NBF5um5um83%否否3320ng105ng穿刺组织>90%1062.核酸提取样本制备核酸抽提文库制备模板制备测序数据分析FFPESlide

orCurlDeparaffinization

and

PKdigestionAddethanolLysisSave

flow-throughDNARNAStep

4:RecoverRNA

from

flow-throughStep

5:RecoverDNA

from

filtercartridgeStep

1:Deparaffinize

FFPEsectionsorslidesStep

2:ProteaseDigestionStep

3:NucleicAcid

Binding72.1核酸提取的质控之Qubit定量法82.2核酸的质控之电泳判别法DNA总量>100ng30-100ng10-30ng<10ng检测方法电泳20ng检测降解程度电泳10ng检测降解程度，剩余样本真空浓缩至8ul左右不电泳，真空浓缩至8ul左右不电泳，真空浓缩至8ul左右DNA电泳检测降解程度评判A（合格）主条带位置弥散带主要分布区域大于7500bp大于7500bp600-7500bp500bp左右B（合格）C（风险）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

提取总量>30ng提取合格与否结合电泳评判合格（A,B）、风险（C）、不合格（D）10-30ng<10ng未电泳，风险未电泳，不合格92.3核酸提取的质控之Agilent

2100法103.文库构建样本制备核酸抽提文库制备模板制备测序数据分析Genomic

DNA超高多重PCR技术Ion

AmpliSeqShearDNAFragmentsLigation

&Nick-repairSizeSelect+AAOrP1P1StandardAdaptersBarcodeAdapters113.1构建好的文库进行质控质控方法试剂盒/方法灵敏度•

gDNA

checkE-Gel®

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/μLAmplification

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chip(sheared

gDNA

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ng

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bp-7kb)10

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-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构建好的文库质控之Equalizer法2)

抓取3)

洗脱1)

扩增134.模板制备样本准备核酸抽提文库制备模板制备测序数据分析PrimersdNTPs扩增PolymeraseMgCl21

10

0油包水PCR14Cycle1,

AnnealingCycle1,ExtensionCycle2,DenatureCycle2,

AnnealingPolymeraseTemplateCycle2,ExtensionCycle3,DenatureCycle3,

AnnealingCycle3,ExtensionCycle4,DenatureCycle

4,

AnnealingISPCycle4,ExtensionAnd

soon…PrimerOilAqueous154.测序样本准备核酸抽提文库制备模板制备测序数据分析测序引物退火测序酶结合珠子落入孔内上机测序16测序原理dNTP•

四种核酸分子依次流过Ion

Chip™

(半导体芯片)•

每个小孔均有一个传感器记录每次测序反应的信号•

直接检测自然的DNA延伸过程H+•

单张芯片每次检测百万计的测序反应∆pH∆QSensingLayerSensor

Plate∆VBulkDrainSilicon

SubstrateSourceTo

columnreceiver17T

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Sphere™Key

Sequence

Sequence

ofInterestAndsoon…21TTTAAKey

SequenceT

C

AG22测序结果的质控参数%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测序结果的质控参数测序通量符合芯片规格要求Navigation

barBarcodingtable(if

applicable)Unaligned/Aligned

metrics推荐测序通量Chip

TypeIon314v2BCIon316v2BCIon318v2BCIon520TotalReads≥400,000Plugin

summariesOutput

files≥2,000,000≥4,000,000≥4,000,000≥15,000,000≥60,000,000Ion530Ion54024测序结果的质控参数测序片段读长符合该panel的amplicon实际长度•

样品测序深度（Mean

Depth）符合实验设计>2000•

在靶率（On

Target）>90%

*•

扩增子均一度（Uniformity）>90%

**FFPE样品根据样品DNA质量状态，相应数据可能会低于新鲜组织样品255.数据分析样本制备核酸提取文库制备模板制备测序数据分析TorrentServerIonReporter或其它分析平台设置运行模式运行初级数据分析次级数据分析深度分析IonTorrent™PGM™

SequencerTorrent

Browserand

PluginsTorrent

ServerTorrent

ServerRunAssessmentAlignmentsQualityScoresVariantCallsDataDelivery(BAM)To

BasesTorrent

Suite™

SoftwareRunAnalysis26数据分析结果AlleleSourceChrom

PositionRefVariantAlleleCallFrequency

TypeAlleleNameGene

IDchr9

80409443chr9

80409448chr17

7577559chr17

7577558chr17

7577559chr17

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