MiSeq高通量测序在临床的应用介绍ppt课件

,MiSeq,高,通量测,序,在临床,的,应用介,绍,Illu,m,ina,MiSeqIllumina,1,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,Library,Preparation,Data,Analysis,Cluster,Generation,Sequenci,n,g,文库制,备,：,处理,样,品,，,加,接,头,和,样,本,特,异性,标签，,为上,机做,准,备,簇生成：,信号,放大的,过,程,2,测序,数据处理,Illumina,新,一代测序流,程,COMPANY CONFIDENTIAL FOR INT,2,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,文库制,备,-,决定测序成,功,与,否,的,关键,步,骤,dual Index,Library,shown,文库,制备,的,目的,是,在需,要,测序,的,DNA,片,段,两,端加,上,能够,与,测序仪,配合,的,接,头,序,列,（,Multiplexed,SR,PE,）,3,双,端标,签,文库,与流动,槽（,Flow,Cell,）,结合,的区域,Read,1,和,Read2,测序引物结合的区域,插入片段,标签序,列区域（,Index,）,COMPANY CONFIDENTIAL FOR INT,3,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,What is a Flow,Cell?,流动槽,(Flow,Cell),是什么,?,A flow cell is a thick glass slide,with,channels or lanes,一,种含,有,通道的,厚,玻璃片,Cluster generation occurs on a flow cell,簇生成在流,动,槽,（,flow,cell,）,上,完,成,Each lane is randomly coated with a,lawn,of oligos that are complementary to library adapters,每,条,通,道,中,都,随,机,植,入,了,能,与,文,库,接,头,互补,结,合的大,量,短DNA片段,4,COMPANY CONFIDENTIAL FOR INT,4,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,簇,生成过,程,接头序列,3,extension,3,延伸,流动槽,表,面随 机植入,了,大量,引物链,片,段杂,交,与延伸,新合成链,双链,DN,A,变性,原始模,板,链,Discard,洗,去,丢弃,单链,DNA,桥式,PC,R,扩增,双链,DNA,桥式,结,构,变性,桥式,PC,R,扩增,5,COMPANY CONFIDENTIAL FOR INT,5,6,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,Illumina,高通量测序原,理,准确的边合成边测序技术,Add 4,Fl-NTPs,+,Polymerase,加入,4,种,不同,荧,光标,记的,dNTP,和,DNA,合成酶,Incorporated,FI-NTP imaged,对结合,上,的荧光,dNTP,进行,照相,Terminator,&,fluorescent,dye,cleaved,from,FI-NTP,结合,在,DNA,链上,的,荧,光,NTP,中 的荧光,标,记和,阻,断基,团,被切,除,，,可以继,续,下一,轮,反应,X 36-,301,6COMPANY CONFIDENTIAL FOR IN,6,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,1,00,Microns,7,Clust,e,rs,DNA,簇,COMPANY CONFIDENTIAL FOR INT,7,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,1,2,3,7,8,9,4,5,6,T,T,T,T,T,T,T,G,T,T,G,C,T,A,C,G,A,T,The,identity of,each,base of,a,cluster,is read,off,from,sequential,images,根,据每,个,点,每,轮,反,应读,取,的,荧,光,信,号序,列,，,转,换,成,相应,的,DNA,序列,8,照,相读取,序,列,COMPANY CONFIDENTIAL FOR INT,8,9,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,i5,Index,Seq Primer,(SBS3rev/BP13),i5,Index,Index,Read 2,(i5),i7,Index,1,2,3,Pa,i,r,ed End around,Tu,r,n,4,1,2,3,Pa,i,r,ed,-,End Tu,r,naround,4,双,index,文库,MiSeq,HiSeq 2500,NovaSeq,MiniSeq,NextSeq550,HiSeq,3k/4k/Xten,Dual indexed sequencing utilizes 4 sequencing,reads,9COMPANY CONFIDENTIAL FOR IN,9,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,BaseSpace,提供完整,的,数,据,存,储和,分,析功,能,Step,1:,Obtain,Results,数据,可以,实,时上传,Step,3:,Annotate/Filter,注释,及,时更新,Step,2:,Align/Call,Variants,分析,即,点即用,BWA,Enrichment,Processes,WGS,reads using BWA,for,alignment,Uses,GATK,for,variant,detection,Enrichment,Performs,read mapping using,Isaac,Offers 4x faster,alignment with,same,accuracy,as BWA;Designed by,Illumina,Enables extraction,of,biological knowledge,from,variant data by providing a rich collection,of,annotation databases,flexible filtering,and a streamlined variant,classification,and reporting,tool,10,COMPANY CONFIDENTIAL FOR INT,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,Illumina,主要测序平台,介,绍,Decreasing price per,GB,Increasing,system,output,For,Research Use,Only.,Not,for,use in diagnostic,procedures.,15 Gb,|,25M,2x300,Mi,S,eq,7.5,Gb,|,25M,2x150,MiniS,e,q,120 Gb,|,400M,2x150,NextSeq550,167,6000 Gb,|1.6,20B,2x150,Mid,High,Micro,Nano V2,V3,Mid,High,S1,S2,S4,NovaSeq,6000,桌,面式,测,序,仪,HiSeq,X,1300-1800Gb|5.3-6B,2x150,生,产级,测,序,仪,1.2,Gb,|,4M 2x150,11,i,S,eq,COMPANY CONFIDENTIAL FOR INT,12,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,.,最新型台式,测,序,平,台,iSeq,靶,向测,序,Choose from,fixed,or custom,panels,Amplicon,DNA,and,RNA,available,小,基因,组,测,序,Sequence,small,genomes,with,ease,辅,助性,应,用,Off-the-shelf,optimized,solutions,Intuitive workflows,for fast,implementation,2x150,base,pairs,1.2,gigabases,4M,reads,917,hour run,time,12COMPANY CONFIDENTIAL FOR I,12,COMPANY,CON,13,FIDENTIAL FOR INTERNAL USE,ONLY,MiSeq,的优点,长度,长,support,application,breadth,多种,测序,试,剂盒,for,flexibility,in,samples and,applications,简单,易用,to,use,workflow,For Research Use,Only.,Not,for,use,in,diagnostic,procedures.,COMPANY CONFIDENTIAL FOR INT,13,C,O,M,PAN,Y CONFIDENTIAL FOR INTERNAL USE,ONLY,MiSeq,System,PE300,|,75%,Q30,0.3,15,|,gigabases,1,25,million,|,clusters,For Research Use,Only.,Not,for,use,in,diagnostic,procedures.,14,COMPANY CONFIDENTIAL FOR INT,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,MiSeq,提供多,种,reads,数的试,剂盒,支,持,不,同应,用,M,i,Seq,Core,Consumables,Version,3,600,cycles,150,cycles,Core,Consumables,Version,2,Micro,300,cycles,(Micro),1 Million,reads,4,Million,reads15 Million,reads,25 Million,reads,Core,Consumables,Version,2,Nano,500,cycles,(Nano),300 cycles,(Nano),Core,Consumables,Version,2,500,cycles,300,cycles,150,cycles,For Research Use,Only.,Not,for,use,in,diagnostic,procedures.,15,COMPANY CONFIDENTIAL FOR INT,COMPANY,CONFIDENTIAL FOR INTERNAL USE,ONLY,Chemistry,Version,Number,of,Reads,(mil),Cycles,Output(Gb),Sequencing,Time,(hrs),3,25,600,15,56,3,25,150,3.8,21,2,15,500,8.5,39,2,15,300,5.1,24,2,15,50,0.85,5.5,2,4,(Micro),300,1.2,28,2,1,(Nano),500,0.5,19,2,1,(Nano),300,0.3,19,MiSeq,提供多,种,reads,数的试,剂盒,支,持,不,同应,用,For Research Use,Only.,Not,for,use,in,diagnost