高通量測序技術(shù)及原理介紹課件

1、高通量測序技術(shù)及原理介紹童貽剛 軍事醫(yī)學(xué)科學(xué)院 微生物流行病研究所 tong.yigang 公司系統(tǒng)名測序長度優(yōu)點缺點Roche/454 FLX System200_700讀長最長；通量高同聚性錯誤；儀器和試劑價格貴Illumina HiSeq 2000/miSeq2 x 150 通量非常高價格貴；后期分析復(fù)雜ABI/SOLiD 5500 xl SOLiD25_35 通量高；試劑消耗少讀長太短Helicos HeliScope 25_30 通量高讀長太短1415測序平臺測序長度進化過程產(chǎn)出測序時間SOLiD30bp15bp30G10天Solexa Hiseq2000150bp X 230bp，

2、50bp，75bp，100bp600G14天454750bp100bp，400bp0.7G7小時37301000bp X 96300bp，600bp0.0001G2小時Illumina workflowSample preparationShearing, ligate adapterCluster generationBridge PCRSequencing on Genome Analyzer IIxRTA (Run Time Analysis) Analysis pipelineOffline analysis, alignment, SNPs calling, reads counti

3、ngVisualize the data, reports the resultsSequencing processFragment DNARepair ends / Add A overhangLigate adaptersSelect ligated DNAHybridize to flow cellExtend hybridized oligosPerform bridge amplificationPerform sequencing on forward strandRe-generate reverse strandPerform sequencing on reverse st

4、randCONFIDENTIAL DO NOT DISTRIBUTE1 Library prep ( 6 hrs)2 Automated Cluster Generation ( 5 hrs)1-8 samples3 Sequencing ( 46 to 120 hrs)1-8 samplesSample Prep - ResequencingSurface bound adapter 1Sequencing primer binding siteSurface bound adapter 2CONFIDENTIAL DO NOT DISTRIBUTECONFIDENTIAL DO NOT D

5、ISTRIBUTE Clonal clusters aregenerated in a containedenvironment (need noclean rooms) Sequencing alsoperformed in the flow cellon the generated clustersFlow cell8 channelsKey to the simplifiedworkflowSurface of flowcell coatedwith a lawn ofoligo pairsCluster generation:Hybridize fragment & extendAda

6、ptersequence 50 M singlemoleculeshybridize to thelawn of primersBound moleculesare then extendedby polymerases3 extensionCONFIDENTIAL DO NOT DISTRIBUTEDouble-strandedmolecule isdenatured.Original templateis washed away.Newly synthesizedcovalentlyattached to theflow cell surface.CONFIDENTIAL DO NOT D

7、ISTRIBUTECluster generation:Denature double-stranded DNANewlysynthesizedstrandOriginaltemplatediscardCluster generation: Covalently boundspatially separated single moleculesSinglemoleculesbound toflow cell ina randompatternCONFIDENTIAL DO NOT DISTRIBUTECluster generation: Bridge amplificationSingle-

8、strand flipsover to hybridize toadjacent primers toform a bridge.Hybridized primeris extended bypolymerases.CONFIDENTIAL DO NOT DISTRIBUTECluster generation: Bridge amplificationdouble-strandedbridge is formed.CONFIDENTIAL DO NOT DISTRIBUTECluster generation: Bridge amplificationDouble-stranded brid

9、geis denatured.Result: Two copies ofcovalently bound single-stranded templates.CONFIDENTIAL DO NOT DISTRIBUTECluster generation: Bridge amplificationSingle-strands flip overto hybridize to adjacentprimers to form bridges.Hybridized primer isextended by polymerase.CONFIDENTIAL DO NOT DISTRIBUTECluste

10、r generation: Bridge amplificationBridge amplificationcycle repeated tillmultiple bridgesare formedCONFIDENTIAL DO NOT DISTRIBUTECluster generationdsDNAbridgesdenatured.Reversestrandscleavedandwashedaway.CONFIDENTIAL DO NOT DISTRIBUTECluster generation leavinga clusterwith forwardstrands only.CONFID

11、ENTIAL DO NOT DISTRIBUTECluster generationFree 3 endsare blocked topreventunwantedDNA priming.CONFIDENTIAL DO NOT DISTRIBUTECONFIDENTIAL DO NOT DISTRIBUTEhybridizedto adaptersequence.SequencingSequencingprimer isSequencingprimerAdd 4 Fl-NTPs +PolymeraseIncorporatedFl-NTP isimagedTerminator andfluore

12、scent dyeare cleaved fromthe Fl-NTPX 36CONFIDENTIAL DO NOT DISTRIBUTESequencing primerFlow cell imagingTotal Internal Reflection FluorescenceFluidics portFlow cellPrismFluidics portCONFIDENTIAL DO NOT DISTRIBUTECONFIDENTIAL DO NOT DISTRIBUTEPaired end sequencingSequencedstrandstripped off3-endsunblo

13、ckedPaired end sequencingBridgeformation3extensionCONFIDENTIAL DO NOT DISTRIBUTEPaired end sequencingDoublestrandedDNA isdenaturedCONFIDENTIAL DO NOT DISTRIBUTEPaired end sequencing3 endsareblockedOriginalforwardstrand iscleavedCONFIDENTIAL DO NOT DISTRIBUTEAdd 4 Fl-NTPs +PolymeraseIncorporatedFl-NT

14、P isimagedTerminator andfluorescent dyeare cleaved fromthe Fl-NTPX 36 - 50CONFIDENTIAL DO NOT DISTRIBUTESequencing reverse strandHybridizesequencingprimerSolexaFlow cell in GAIIxCONFIDENTIAL DO NOT DISTRIBUTEImage re-analysis piplelineImageAnalysisBasecallingSequenceAnalysisGA Analysis PipelineInstr

15、ument PCAnalysis PC/clusterdatatransferImages (.tif)Lane 1.8Cycle 1.36Tile_Cycle_Image_a,Tile_Cycle_Image_c,Tile_Cycle_Image_g,Tile_Cycle_Image_t.params fileFor each tile:Cluster intensitiesCluster noiseFor each tile:Corrected cluster intensitiesCluster sequenceCluster probabilitiesFor all data:Qual

16、ity FilteringSequence AlignmentRun Statistics VisualizationCONFIDENTIAL DO NOT DISTRIBUTEBustardBase with highest corrected intensity is calledACGTCGeraldI AI A + IBGEneration ofRecursiveAnalysesLinked byDependencyIAIBFiltering removes low quality base callsChastity: C =Default value 0.6Other filter

17、s include purity,similarity, neighbor andneighborhood.CONFIDENTIAL DO NOT DISTRIBUTEBustard output *_qseq.txtMachine nameRun number Lane number Tile number X coordY coordSequence Quality PassedFilter IndexRead formatEAS1 89 1 59 111 525 AACCTT 2 TGACCAGCGTCAACCAGTACTACGTCTTTGTCGATAG aaaaa_V_OYOZZYUP

18、JZRX 1EAS1 89 1 59 111 726 AACCTT 2 TCTGGATGAAGAACGATCCGCTGCAGAGGTGCTGGCA _FNXXZWFZ_YYTYMUVBBBBBBBBBBB 0EAS1 89 1 59 111 860 AACCTT 2 TATCGCGTAGTGTAGCACGGCCTTTTTTTCGTCCACC aaaXFUWQUHVN_ZRWZZXFWYFTX 1EAS1 89 1 59 112 377 AACCTT 2 TTTTCTTCTCCTTCGCCATCAGCGACAAAATCAAGCA abbbabbbbbbaaaTaaaaaY_YNaZZ 1EAS1

19、 89 1 59 112 538 AACCTT 2 TGTGAATTAACAGTATTGGCGTAGTTACAGGCAGTGT aa_aabbaaa_aSYZYUBBBBB 1EAS1 89 1 59 112 576 AACCTT 2 TCTCCTTCGTCTTCTTCCATCAGTTGTTCGACCGGCT GJRNGBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 0EAS1 89 1 59 112 607 AACCTT 2 TCCACCATCAACTGGTTGCCAGTGCGCGGGCAGTTAA aabaaaaaaX_YTTHTTZQYTX 1EAS1 89 1 59 112

20、 255 AACCTT 2 TGATGCTGATAAGCAGCGTGCTCACAACCCAGATTTG aaba_abaabbbbabababbbbb_aba_Zabbb 1Fastq formathttp:/www.bioinformatics.babraham.ac.uk/projects/fastqc/ GERALD sequenceSummary.html(PF: pass filter)FastQCThird part softwareBrief Bioinform. 2011 Jan 18NGS 技術(shù)論壇SEQwiki:http:/NGS軟件大全http:/wiki/Software/list（熒光基團漂白）捕獲技術(shù)Paired-end vs. Mate-PairSOLiD Syst