上海市臨檢中心 二代測序NGS 測序儀的基本操作流程和注意事項

測序儀的基本操作流程和注意事項劉媛

博士Illumina?

2016

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HiSeq,

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MiSeq,

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respectiveowners.illumina引領(lǐng)高通量測序行業(yè)的發(fā)展HiSeq3000/4000NovaSeq的發(fā)布使我們離100美金人基因組測序更近一步重新定NextSeq550增加芯掃描功能義了生產(chǎn)級測序首臺且唯一一臺高通公司成立，總部位量測序系統(tǒng)Miseq片于加州圣地亞哥市，主要業(yè)務(wù)為芯片Hiseq2000上市，高通量測序平臺黃金標準獲美國FDA認證DxHiSeqXFIVEHiseqXTen上市,公司上市，登陸NASDAQ證券Hiseq2500登交易市場1000美金人宣告陸市場,全面加基因組實現(xiàn)Nextseq500市速測序進程桌上型高通量測MiniSeq上市，便收購Solexa公司，首臺高通量測序平臺GA登陸市場場，捷強大的臺式測序儀從此變得觸手可及。更廣泛的通量范圍，適合最廣泛的應(yīng)用序系統(tǒng)Miseq登陸市場1998

2000

200720142010

2011

2012

20132015

2016

2017>4,500

雇員>1200名研發(fā)人員>400技術(shù)支持人員推動全球測序技術(shù)發(fā)展，數(shù)據(jù)結(jié)果接受廣泛>90%of

theworld’s

sequencingdataisgenerated

usingIlluminaSBS

technology.*>90%的測序數(shù)由illumina平臺產(chǎn)出*Datacalculations

onfile.Illumina,Inc.,

2015.3OurVisionToimprove

human

health

byunlocking

the

power

of

the

genome通過解碼基因組的威力來改善人類健康4Illumina高通量基因檢測平臺解決方案SEQUENCINGSYSTEMS

|

Sequencing

bySynthesis

(SBS)POPULATION

POWERPRODUCTION

POWERFLEXIBLE

POWERNextSeq?

550/550DxHiSeq?

4000HiSeq3000FOCUSEDPOWERMiSeq?NovaSeqHiSeq2500MiSeqDx?For

invitro

diagnosticuseARRAY

SCANNERS

|

BeadArray,

Infinium,GoldenGate,

DASLMiniSeq?POWERFUL

ARRAYSCANNERCUTTING-EDGE

ARRAY

SCANNERHiScan?iScan?iSeq

100For

ResearchUseOnly.Notfor

useindiagnosticprocedures.5Illumina提供完整的試劑盒制備方案RRptipDmeReIllumina-AmpliSeq

PanelTruSightPanelsTruSeq

PCR-FreeTruSeq

NanoNexteraTruSeq

RNATruSeq

Stranded

mRNATruSeq/Nextera

ExomeTruSeq

Stranded

TotalRNATruSeq/Nextera-xGenExomeNextera

XTTruSeq

Small

RNATruSeq

Targeted

RNATruSeq

ChIPNextera

DNA

FLexNextera

Mate

PairTruSeq

SyntheticLongRead*6Illumina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：Cluster

GenerationSequencing信號放大的過程?

測序?

數(shù)據(jù)處理Data

Analysis7文庫制備

---

決定測序成功與否的關(guān)鍵步驟①④②③④①②雙端標簽文庫dual

IndexLibrary

shown①

與流動槽（FlowCell）結(jié)合的區(qū)域②

Read1和Read2測序引物結(jié)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DN

片段兩端加上能夠與測序儀配合的接頭序列（Multiplexed,

SR,

P

）8WhatisaFlowCell?流動槽(Flow

Cell)是什么?A

flowcellisathickglass

slidewith

channelsorlanes一種含有通道的厚玻璃片Cluster

generation

occursonaflow

cell簇生成在流動槽（flowce

）上完成Eachlaneisrandomly

coatedwith

alawn

ofoligos

thatarecomplementary

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

片段9簇生成過程雙鏈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擴增雙鏈

橋式結(jié)構(gòu)變性DNA橋式PCR擴增10Illumina高通量測序原理——準確的邊合成邊測序技術(shù)Add

4Fl-NTP’s

+Incorporated

FI-Terminator

&fluorescent

dyecleavedfrom

FI-NTP結(jié)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(yīng)Polymerase加入4種不同熒光標記的dNTP和DNA合成酶NTP

imaged對結(jié)合上的熒光dNTP進行照相X36-30111Clusters

DNA簇100Microns12照相讀取序列T

GC

T

A

CG

A

T

…125678934T

T

T

T

T

T

T

G

T

…Theidentity

ofeachbase

ofa

cluster

isreadofffrom

sequential

images根據(jù)每個點每輪反應(yīng)讀取的熒光信號序列，轉(zhuǎn)換成相應(yīng)的DNA序列13雙index文庫測序過程MiSeq

,HiSeq

2500,

NovaSeq1234Paire

EndTurnaroundMiniSeq,

NextSeq550,

HiSeq

3k/4k/Xten1234PairedEndTurnaroundDualindexed

sequencing

tilizes

4

equencing

eads14BaseSpace

提供完整的數(shù)據(jù)存儲和分析功能Step1:Obtain

Results

數(shù)據(jù)可以實時上傳Step2:Align/CallVariants分析即點即用BWA

Enrichment?

ProcessesWGSreads

usingBWA

foralignment?

Uses

GATK

forvariantdetectionEnrichment?

PerformsreadmappingusingIsaac?

Offers

4xfaster

alignment

with

sameaccuracyasBWA;

Designed

byIlluminaStep3:Annotate/Filter注釋及時更新?

Enablesextraction

of

biological

knowledge

fromvariantdata

byprovidingarich

collectionofannotation

databases,

flexiblefiltering,anda

streamlinedvariantclassification

and

reportingtool15NGS參數(shù)?

由測序平臺和不同試劑盒共同決定?

Amplicom文庫測序時需要讀通測序長度?

Miseq讀長最長有2x301和2X251?

其他儀器最長都是2x151(除hiseq2500)?

測序時讀取到的DNA序列?

衡量轉(zhuǎn)錄組測序時使用reads數(shù)?

測序數(shù)據(jù)量=測序長度Xreads數(shù)Reads?

測序時讀取到的DNA序列?

測序得到的堿基數(shù)量與待測基因組的比值，假設(shè)一個基因大小?

是2M,測序深度為10X,那么獲得的總數(shù)據(jù)量是20M。測序深度Depth?測序獲得的序列占整個基因組的比例，例如某個基因組測序，覆蓋度是98%，測序覆蓋度

?

那么還有2%的序列區(qū)域在本次測序中沒有獲得，這些未覆蓋到的序列一般稱為Gap（往往是由于高GC，重復(fù)序列等原因?qū)е碌摹?Coverage注：1人WGS測序要求30xdepth；2WES一般要求至少80%c