南寧無創(chuàng)產前檢測(NIPT)會議課件 新一代測序技術在臨床檢測中的應用

新一代測序技術在臨床檢測中的應用熊麗

PhD.

MD臨床應用,

大中華區(qū)lxiong@(熊麗）+8618680535035ForResearch

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容Illumina新一代測序技術原理介紹新一代測序技術在臨床的應用ForResearch

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InDiagnosticProcedures.Illumina新一代測序技術原理介紹ForResearch

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InDiagnosticProcedures.新一代測序技術的發(fā)展歷程BGI500PGMIon

Proton停產454停產SolidIlluminaSBSHiSeq

200010K

genomeMiSeqHiSeq2500HiSeq

XNextSeqMiniSeqNovaSeq6000GA1K

genome年份2005年2007年2010年2012年2014年2016年2017年ForResearch

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InDiagnosticProcedures.llumina新一代測序流程?

文庫制備：Library

Preparation處理樣品，加接頭和樣本特異性標簽，

為上機做準備?

簇生成：ClusterGenerationSequencing信號放大的過程?

測序?

數據處理Data

AnalysisForResearch

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InDiagnosticProcedures.文庫制備

---決定測序成功與否的關鍵步驟①④②③④①②雙端標簽文庫dualIndexLibrary

shown①

與流動槽（Flow

Cell）結合的區(qū)域②

Read1和Read2測序引物結合的區(qū)域③

插入片段④

標簽序列區(qū)域（Index）文庫制備的目的是在需要測序的DNA片段兩端加上能夠與測序儀配合的接頭序列（Multiplexed,

SR,PE）ForResearch

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aflow

cell簇生成在流動槽（flow

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InDiagnosticProcedures.生成過程雙鏈DNA變性片段雜交與延伸單鏈DNA接頭序列新合成鏈原始模板鏈Discard

洗去丟棄流動槽表面隨機植入了大量引物鏈3’extension3’

延伸橋式PCR擴增式PCR擴增雙鏈DNA橋式結構變性ForResearch

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InDiagnosticProcedures.Sequencing

BySynthesis邊合成邊測序（SBS）Add

4Fl-NTP’s+

Polymerase加入4種不同熒光標記的dNTP和DNA合成酶Incorporated

FI-Terminator

&fluorescentdye

cleavedfrom

FI-NTP結合在DNA鏈上的熒光NTP中的熒光標記和阻斷基團被切除，可以繼續(xù)下一輪反應NTP

imaged對結合上的熒光dNTP進行照相X

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InDiagnosticProcedures.usters

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images根據每個點每輪反應讀取的熒光信號序列，轉換成相應的DNA序列ForResearch

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InDiagnosticProcedures.新一代測序技術在臨床的應用ForResearch

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InDiagnosticProcedures.uminaNext

Generation

Sequencing

PlatformS1,S2,S3,S4NovaSeq

6000Low,

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Gb

|1.6

–20B2x150NextSeqMicro,NanoV2,

V3Mid,

HighMiSeqMiniSeq120

Gb|

400M2x15015

Gb|25M2x3007.5

Gb

|25M2x150Decreasing

price

perGBForResearch

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InDiagnosticProcedures.llumina

高通量測序覆蓋各個領域生殖健康農業(yè)腫瘤生物制藥遺傳病感染性疾病法醫(yī)學健康篩查科學研究人類健康ForResearch

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InDiagnosticProcedures.llumina高通量檢測技術推動出生缺陷防控一級預防攜帶者篩查植入前遺傳學篩查三級預防二級預防產前篩查/產前診斷遺傳病檢測新生兒篩查24sureNIPT24sure+靶向測序CytoSNPKaryomappingNGS

NextSeq臨床外顯子組測序外顯子組測序CytoSNP-12CMAVeriSeqPGSCytoSNP-850KPanel靶向測序臨床外顯子組測序外顯子組測序NGS

NextSeqForResearch

UseOnly.

NotForUseInDiagnosticProcedures.胚胎植入前遺傳學篩查(PGS)/胚胎植入前遺傳學診斷(PGD)PGS胚胎染色體異常檢測MiSeq/MiSeq

Dx測序染色體易位或倒位形成的片段異常檢測PGD基因芯片判定染色體病PGDNextSeq550測序/芯片iScan芯片系統(tǒng)Karyomapping核型定位判定單基因病ForResearch

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InDiagnosticProcedures.二級防控–

早發(fā)現、早干預減少出生缺陷兒的出生一級預防三級預防二級預防攜帶者篩查植入前遺傳學篩查產前篩查/產前診斷遺傳病檢測新生兒篩查NIPTCytoSNP-12CMACytoSNP-850K靶向測序臨床外顯子組測序外顯子組測序NGS

NextSeqForResearch

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InDiagnosticProcedures.常見可出生的三體綜合征T21T18T13Other

rare16%Sextrisomy5%45,X8%T2153%T135%T1813%唐氏綜合征愛德華綜合征帕陶綜合征孕期常見的胎兒染色體異常21

三體18

三體13

三體Source:

Data

adaptedfrom

Wellesley,

D,

et

al.,Rarechromosome

abnormalities,

prevalenceand

prenatal

diagnosisrates

from

population-based

congenitalanomalyregisters

inEurope.

EurJof

HumGen

11

January

201218ForResearch

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InDiagnosticProcedures.女性年齡的影響15%10%5%1:81:5261:16671:110%20304050女性年齡唐氏綜合征妊娠風險其它染色體異常的妊娠風險Source:

GraceMR,

etal.

Obstet

Gynecol

Surv.

2016

Aug;71(8):477-87ForResearch

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InDiagnosticProcedures.傳統(tǒng)產前篩查三體的檢測率檢測率

(%)B超監(jiān)控(NT值)

或

血清學篩查(PAPPA+HCG)早孕期早孕期64-70孕早期血清學篩查B超監(jiān)控(NT值)82-8769血清學三聯篩查(AFP+HCG+uE3+inhibition)中孕期81中孕期血清學四聯篩查

(AFP+HCG+uE3+inhibition)聯合篩查94-9685-88孕早期血清學篩查B超監(jiān)控孕中期血清學篩查血清學聯合篩查孕早期血清學篩查孕中期血清學篩查誤判率:

5%Source:

ACOG

PracticeBulletin

No.77,January

2007ForResearch

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NotForUse

InDiagnosticProcedures.無創(chuàng)產前篩查on-invasiveprenataltesing(NIPT)細胞凋亡釋放cell-free

DNA(cfDNA)–

通過DNA片段的形式釋放入母血循環(huán)–

母體血液

cf

DNA中有2–20%來源于胎兒(來自于胎盤)理想的妊娠期非整倍體分析物–

妊娠9周以上即可被檢出–

產后數小時內清除NIPT取材無需侵入性手段ForResearch

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InDiagnosticProcedures.NIPT的技術類型基于新一代測序的NIPT基于芯片的NIPT全基因組測序靶向測序SNP-芯片大規(guī)模并行測序(MPS)單核苷酸多態(tài)性(SNP)非-SNPForResearch

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InDiagnosticProcedures.全基因組測序NIPT原理提取及制備cfDNA新一代測序(NGS)12NextSeq

CN500國械注準20153400460NextSeq

550AR國械注準20173400330ForResearch

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InDiagnosticProcedures.全基因組測序NIPT原理對特定的

cfDNA

測序子3

配對Count:AGGTACCGATGACTTCCAGGCGATTTAACT…ACCACGATTTAACTGGAGTAAAGACTTCCAGGTACCGATCTAGCCT…

人類基因組序列每個樣本可獲得數以百萬計的“counts”ForResearch

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InDiagnosticProcedures.算胎兒非整倍體胎兒cfDNA(20%)4

計數21號染色體的cfDNA

超出10%（21三體樣本）母體cfDNAVSZ-Score……染色體:12321Trisomy

21ForResearch

UseOnly.

NotForUse

InDiagnosticProcedures.于全基因組測序的NIPT

(MPS-NIPT)提供精確的、全基因組范圍的檢測12313X414Y567891020112112221516171819全染色體覆蓋優(yōu)勢–

相對其他NIPT技術檢測失敗率最低

(<1.4%1,2)–

分析方式成熟快速

(樣本到結果的時間快)–

檢測范圍可擴展Reference:1.

Bhatt

et

al.Poster

presented

at

ISPD,20142.

Palomaki

GE,

et

al.Genet

Med.2012Mar;14(3):296–305

andmarketed

dataForResearch

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InDiagnosticProcedures.IPT的檢出率enn

P,Borrell

A,Chiu

RW,

etal.Position

statementfrom

the

Chromosome

Abnormality

Screening

theommitteeon

behalf

of

the

Board

of

the

International

Society

for

Prenatal

Diagnosis.

Prenat

Diagn.

2015

Aug;35(8):725-34.ForResearch

UseOnly.

NotForUse

InDiagnosticProcedures.統(tǒng)產前篩查

vs.

NIPT誤診率=5.0%誤診率<0.1%tNIPTACOG

Practice

Bulletin

No.77.

Obstet

Gynecol2007;109:217-27.28ForResearch

UseOnly.

NotForUse

InDiagnosticProcedures.NIPT檢測的挑戰(zhàn)檢測失敗

(no-call

results)假陽性/假陰性結果雙胎妊娠ForResearch

UseOnly.

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InDiagnosticProcedures.2016Offering

diagnostic

testingforano-call

NIPSresult

due

to

low

fetalfractionif

maternal

blood

for

NIPSwasdrawn

at

anappropriate

gestational

age.

Arepeat

blood

drawisNOTappropriate.當在適合進行NIPS的孕周采血進行檢測時出現因胎兒游離DNA含量低導致的no-call結果，建議進一步行診斷性檢測，不建議重采血進行NIPS檢測Offeringaneuploidy

screening

other

thanNIPSincasesof

significant

obesity.對于顯著肥胖孕婦，建議進行傳統(tǒng)非整倍體篩查而不推薦NIPS。Alllaboratories

should

include

aclearly

visible

fetalfractiononNIPS

reports.檢測實驗室應在NIPS檢測報告上提供胎兒DNA含量信息。Alllaboratories

should

establish

and

monitor

analyticalandclinical

validityforfetalraction.檢測實驗室應建立胎兒DNA含量分析方法并監(jiān)測和驗證檢測效度。Alllaboratories

should

specifythereason

for

a

no-callwhen

reporting

NIPSresults.檢測實驗室如出現NIPS

no-call情況時，應在報告上注明原因ForResearch

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NotForUse

InDiagnosticProcedures.NIPT的挑戰(zhàn)

–造成假陰性/假陽性結果的生物學因素分析1,6嵌合體Generalized

Confinedplacentalmosaicism

mosaicismFetalmosaicism雙胎之一死亡2,3母體存在遺傳變異2,4,5,7母體的疾病狀態(tài)

(e.g.器官移植，腫瘤等)Choi

H,

Lau

TK,

et.

al.

Fetal

aneuploidy

screening

by

maternal

plasma

DNA

sequencing:

'false

positive'

due

to

confined

placental

mosaicism.

Prenat

Diag

2012,

32,

1–3Bianchi

DW,

et

al.

Fetal

sex

chromosome

testing

by

maternal

plasma

DNA

sequencing:

clinical

laboratory

experience

and

biology.

Obstet

Gynecol.2015

Feb;

125(2):

375-82

doi:10.1097/AOG.0000000000000637.Snyder

MW,

et

al.

Copy-number

variation

and

false

positive

prenatal

aneuploidy

screening

results.

N

EnglJ

Med.

2015

Apr

23;372(17):1639-45.

doi:

10.1056/NEJMoa1408408.Bianchi

DW,

et

al.

Noninvasive

Prenatal

Testing

and

Incidental

Detection

of

Occult

Maternal

Malignancies.

JAMA

2015

Jul

14;314(2):162-9.

doi:

10.1001/jama.2015.7120.Osborne

CM,

et

al.

Discordant

noninvasive

prenatal

testing

results

in

a

patient

subsequently

diagnosed

with

metastatic

disease.

Prenat

Diagn.

2013

Jun;33(6):609-11.

doi:

10.1002/pd.4100.Grati

FR,

et

al.

Fetoplacental

mosaicism:

potential

implications

for

false-positive

and

false-negative

noninvasive

prenatal

screening

results.

Genet

Med.

2014

Aug;

16(8):620-4.

doi:

10.1038/gim.2014Bianchi

DW.

Cherchez

la

femme:

maternal

incidental

findings

can

explain

discordant

prenatal

cell-free

DNA

sequencing

results.

Genet

Med.ForResearch

UseOnly.

NotForUse

InDiagnosticProcedures.多國出臺NIPT指南國際專業(yè)協(xié)會發(fā)表NIPT指導意見美國婦產科醫(yī)師學會（

ACOG）和美國母胎醫(yī)學會（SMFM）美國遺傳學咨詢協(xié)會（NSGC）國際婦產科超聲協(xié)會（ISUOG）國際產前診斷協(xié)會（ISPD）加拿大婦產科醫(yī)師協(xié)會（SOGC）美國醫(yī)學遺傳學及基因組學學院（ACMG）意大利母胎醫(yī)學協(xié)會中國《高通量測序產前篩查技術管理規(guī)范（修改稿）》ForResearch

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InDiagnosticProcedures.IPT

國外指南比較ACOG-2015ISPD-2015ACMG-2016合人群高風險及低風險人群，強調低風險人群的PPV低于高風險人群可針對高風險及低風險群，但強調遺傳咨詢的性-不推薦他染色體非整體檢測當有足夠臨床資料說明孕婦存在死胎、胎兒嚴重畸形或存在妊娠不良結局的高風險時染色體檢測-NIPT能夠評價胎兒部分性染色體應用可以擴展到性染色異常及X單體，但NIPT的假陽性率

整倍體篩查尚存在爭議Vs不建議NIPT用于微缺失綜合征篩

可以用于臨床意義明確且有嚴重臨床結局的染色體微缺失/查復綜合征胎不適合雙胎妊娠NIPT篩查效率類似于單胎妊娠，但ISPD強調雙胎受胎兒游離DNA濃度影響其檢測失敗率增加檢測實驗室需要充分評call-不建議重抽血綜合孕婦年齡，胎兒的異常超聲檢測結果后制定下一步檢測計劃mmittee

Opinion

Summary

No.640:

Cell-FreeDNAScreening

For

FetalAneuploidy.Obstet

Gynecol.

2015

Sep;126(3):691.2.doi:

10.1038/gim.2016.97.

Epub2016

Jul

28.egg

AR,Skotko

BG,Benkendorf

JL.Noninvasive

prenatal

screening

forfetaluploidy,

2016

update:

aposition

statement

ofthe

American

College

of

MedicalGenetics

and

Genomics.

Genet

Med.

2016

Oct;18(10):1056-65.Benn

P,BorrellA,ChiuRW,

etal.Position

statement

fromtheChromosome

Abnormality

Screening

thenternational

Society

for

Prenatal

Diagnosis.

Prenat

Diagn.

2015

Aug;35(8):725-34.Committee

onbehalf

of

the

BoardofForResearch

UseOnly.

NotForUse

InDiagnosticProcedures.通量測序產前篩查技術管理規(guī)范（修改稿）適用時間高通量基因測序產前篩查可在孕12+0周后進行，為遵循在孕周滿28周前完成產前篩查和診斷的原則，建議篩查的時限通常為

12+0-22+6周。臨床應用指征高通量測序產前篩查作為一種高精度的產前篩查方法，適用于需精確評估三種常見胎兒染色體疾病風險的孕婦，但對于部分孕婦人群存在慎用或不適用的情況，具體為：ForResearch

UseOnly.

NotForUse

InDiagnosticProcedures.高通量測序產前篩查技術管理規(guī)范（修改稿）慎用人群一

不適用人群核對孕周后篩查時孕周小于12+0周早中孕期產前篩查高風險孕婦預產期年齡達到或超過35歲的高齡孕婦重度肥胖（體重指數BMI>40）孕婦有染色體異常胎兒分娩史，夫婦一方有明確色體異常的孕婦孕婦

1

年內接受過異體輸血、移植手術、細治療或接受過免疫治療等對高通量基因測序前篩查與診斷結果將造成干擾的通過體外受精-胚胎移植（以下簡稱

IVF-ET）胎兒影像學檢查懷疑胎兒有微缺失微重復綜征或其他染色體異?？赡艿姆绞绞茉械脑袐D雙胎及多胎妊娠的孕婦各種基因遺傳病的高風險孕婦人群已知孕期合并惡性腫瘤的孕婦醫(yī)生認為可能有影響篩查準確性的其他情形醫(yī)生認為明顯影響篩查準確性的其他情形ForResearch

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InDiagnosticProcedures.基于NGS的NIPT發(fā)展方向染色體微缺失/微重復綜合征單基因疾病檢測Thalassemia胎兒甲基化測序胎兒轉錄組測序cfDNA漿NGS技術Wong

FC,Lo

YM.

Annu

Rev

Med.

2016;67:419-32.

doi:10.1146/annurev-med-091014-115715.ForResearch

UseOnly.

NotForUse

InDiagnosticProcedures.常見染色體微缺失/微重復綜合征發(fā)生率常見染色體非整倍體異常發(fā)生率缺失綜合征缺失大小發(fā)生率George綜合征~85%>3Mb1/4,00021三體綜合征1/74036微缺失綜合征100

Kb–100Mb5–6

Mb1/4,000–10,0001/10,000–25,0001/12,00018三體綜合征13三體綜合征Tuner

綜合征1/60001/120001/5000der-Willi

綜合征q11.2-q13)gelman

綜合征q11.2-q13）5–6

Mb叫綜合征

(5p-)3.2Mb–30

Mb1.9–30Mb1/20,000–50,0001/50,000lf-Hirschhorn

綜合征-)ForResearch

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InDiagnosticProcedures.無創(chuàng)產前染色體微缺失/微重復檢測WorkflowExpanded

NIPT低覆蓋度的全基因組測序SNP-based靶向測序基于Bin為單位的分析窗CountingZ-scoreForResearch

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InDiagnosticProcedures.無創(chuàng)產前染色體微缺失/微重復檢測N=175393，采用低覆蓋度全基因組測序進行染色體微缺失綜合征進行檢測發(fā)現Digeroge綜合征32例，陽性率0.02%，其中31例確診為陽性，1例可疑陽性，未發(fā)現假陰性性Lyn

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InDiagnosticProcedures.N=1422q11缺失綜合征，胎兒可疑孕婦及胎兒均為22q11患者孕婦為22q11患者孕婦和胎兒均為可疑陽性5例單一胎兒可疑陽性3例當孕婦為染色體微缺失綜合征患者時，檢測的假陽性率增加1例孕婦可疑，胎兒診斷為22q11微缺失綜合征ForResearch

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InDiagnosticProcedures.無創(chuàng)產前染色體微缺失/微重復檢測Total2116113孕婦存在的染色體微缺失增加了NIPT假陽性率Lyn

S.

ChittyandDianaW.

Bianchi，Prenat

Diagn.2015Oct;

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InDiagnosticProcedures.無創(chuàng)產前染色體微缺失/微重復檢測N=21948，基于SNP的靶向測序（672SNPs）進行22q11微缺失綜合征檢測10715進行22q11.2微缺失篩查，11233對包括22q11微缺失綜合征在內的5個的微缺失綜合征進行檢測,陽性率0.5%（95/21948），真陽性11例，其中孕婦為缺失綜合征，50例假陽性，34例結局不清,隨著測序深度增加PPV可提高.

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InDiagnosticProcedures.挑戰(zhàn)和展望假陽性假陰性：22q11缺失2.1M出現假陰性原因：缺失片斷的大小，cffDNA%濃度，測序深度，孕婦的疾病攜帶狀態(tài)發(fā)展方向：對已知致病性，發(fā)生率較高的染色體微缺失綜合征進行無創(chuàng)產前篩查ForResearch

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InDiagnosticProcedures.無創(chuàng)單基因病檢測疾病位點相關聯的SNP位點突變位點檢測MN母親基因型orN胎兒基因型orMcfDNAM>NM<NM=NX-Link

AR/ADRhD

血型鑒定RMD（相對突變劑量檢測）RHDO（相對單倍型劑量檢父源性突變位點檢測Lun

FM,Lo

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InDiagnosticProcedures.無創(chuàng)單基因病檢測策略基于NGS的單基因無創(chuàng)檢測策略全基因組測序靶向測序SNPNon-SNPRMDRHDORHDOForResearch

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InDiagnosticProcedures.挑戰(zhàn)與展望檢測方法優(yōu)點挑戰(zhàn)Non-SNP靶向測序直接針對致病基因突變位點進行檢測1.針對不同單基因疾病時需要不同的解決方案，母源性突變檢測準確率受限2.受同源基序列或假基因影響檢測準確性3.受捕獲方法的效率影響基于SNP的靶向測序

1.針對疾病相關聯的SNP進行連鎖分析，通過

1.間接檢測方法雙親不同基因型的SNPs位點分析，判斷胎兒

2.需要進行家系分析完成檢測的基因型；3.受捕獲方法的效率影響2.可提高母源性突變的檢測準確率3.通過SNPs基因型的分析，可避免同源基因或假基因影響全基因組測序可通過單倍型分析方法對臨床可檢測的單基因

1.基于全基因組的單倍型分析方法存在挑戰(zhàn)病進行檢測2.文庫構建可能會引入新突變，無法區(qū)分胎兒自身的新發(fā)突變ForResearch

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InDiagnosticProcedures.NIPT—胎兒甲基化測序

目前基于SNP和分化的甲基化CpG兩種方法可以用于胎兒甲基化分析；

特定甲基化標記物可用于胎兒非整倍體和CNV篩查；

血液中母體和胎兒甲基化水平可用于特定病篩查。IPT—胎兒轉錄組測序AdaI.C.Wong

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InDiagnosticProcedures.基于新一代測序技術的遺傳病檢測策略靶向測序（疾病目標基因群測序）●對某一類疾病（或表型）已知的致病基因及相關聯的基因進行N全基因組測序臨床外顯子測序基于探針捕獲的靶向測序(百kb~15Mb)（全外或臨床全外）●對已知與臨床表型相關的外顯子區(qū)域進行全面但有針對性的測序基于擴增子的靶向測序(Targets

100s)全外顯子組測序●對人類基因組全部蛋白質編碼區(qū)域（整個外顯子組）的基因集進行高通量測序，可檢測與表型相關聯的已知或未知突變PCR

擴增測序Targets

=10s全基因組測序DNA●對人類全基因組序列進行測序，包含序列外顯子區(qū)和非編碼區(qū)域ForResearch

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