1、MiSeq高高通量测通量测序序在临床在临床的的应用介应用介绍绍IlluminaCOMPANY CONFIDENTIAL FOR INTERNAL USE ONLYLibrary PreparationData AnalysisCluster GenerationSequencing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新新一代测序流一代测序流程程COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY文库制文库制备备 -决定测序成决定测序成功功与与否否的的关键关键步步骤骤dual Index Li
2、braryshown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 与流动与流动槽（槽（Flow Cell）结合结合的区域的区域 Read 1和和Read2测序引物结合的区域测序引物结合的区域 插入片段插入片段 标签序标签序列区域（列区域（Index）COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYWhat is a Flow Cell?流动槽流动槽(Flow Cell)是什么是什么?A flow cell is a thick glass slide with channels
3、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4COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY簇簇生成过生成过程程接头序列接头序列3 extension3 延伸延伸流动槽流动槽表表面随面随 机植入机
4、植入了了大量大量 引物链引物链片段杂交与延伸新合成链双链DNA变性原始模板链Discard 洗洗去去丢弃丢弃单链DNA桥式PCR扩增双链DNA桥式结构变性桥式PCR扩增56COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYIllumina高通量测序原高通量测序原理理准确的边合成边测序技术准确的边合成边测序技术Add 4 Fl-NTPs+Polymerase加入加入4种种不同不同荧荧光标光标 记的记的dNTP和和DNA 合成酶合成酶Incorporated FI-NTP imaged 对结合对结合上上的荧光的荧光 dNTP进行进行照相照相Terminator&f
5、luorescent dye cleaved from FI-NTP结合结合在在DNA链上链上的的荧荧光光NTP中中 的荧光的荧光标标记和记和阻阻断基断基团团被切被切除除，可以继可以继续续下一下一轮轮反应反应X 36-301COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY100 Microns7Clusters DNA簇簇COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY123789456T T T T T T T G T T G C T A C G A T The The identity of identity of e
6、ach each base of base of a a cluster cluster is read is read off off from from sequentialsequential imagesimages根根据每据每个个点点每每轮轮反反应读应读取取的的荧荧光光信信号序号序列列，转转换换成成相应相应的的DNADNA序列序列8照照相读取相读取序序列列9COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYi5 Index Seq Primer(SBS3rev/BP13)i5 IndexIndex Read 2(i5)i7 Index123Paire
7、d End aroundTurn4123Paired-End Turnaround4双双index文库文库MiSeq,HiSeq 2500,NovaSeqMiniSeq,NextSeq550,HiSeq 3k/4k/XtenDual indexed sequencing utilizes 4 sequencing readsCOMPANY CONFIDENTIAL FOR INTERNAL USE ONLYBaseSpace 提供完整提供完整的的数数据据存存储和储和分分析功析功能能Step 1:Obtain Results 数据可以实时上传Step 3:Annotate/Filter注释及时更
8、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 detectionEnrichment Performs read mapping using Isaac Offers 4x faster alignment with same accuracyas BWA;Designed by Illumina Enables extraction of biological knowledge from variant dat
9、a by providing a rich collection of annotation databases,flexible filtering,and a streamlined variant classification and reporting tool10COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYIllumina主要测序平台主要测序平台介介绍绍Decreasing price per GBIncreasing system outputFor Research Use Only.Not for use in diagnostic pr
10、ocedures.15 Gb|25M2x300MiSeq7.5 Gb|25M2x150MiniSeq120 Gb|400M2x150NextSeq550167 6000 Gb|1.6 20B2x150Mid,HighMicro,Nano V2,V3Mid HighS1,S2,S4NovaSeq 6000桌桌面式面式测测序序仪仪HiSeq X1300-1800Gb|5.3-6B2x150生生产级产级测测序序仪仪1.2 Gb|4M 2x15011iSeq12COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY.最新型台式最新型台式测测序序平平台台 iSeq靶靶向测向
11、测序序Choose from fixed or custom panelsAmplicon DNA and RNA available小小基因基因组组测测序序Sequence small genomes with ease辅辅助性助性应应用用Off-the-shelf,optimized solutionsIntuitive workflows for fast implementation2x150base pairs1.2gigabases4Mreads917hour run timeCOMPANY CON13FIDENTIAL FOR INTERNAL USE ONLYMiSeq 的优点
12、的优点长度长度长长 support application breadth多种多种测序测序试试剂盒剂盒for flexibility in samples and applications简单简单易用易用to use workflowFor Research Use Only.Not for use in diagnostic procedures.COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYMiSeq SystemPE300|75%Q300.3 15|gigabases 1 25 million|clustersFor Research Use Onl
13、y.Not for use in diagnostic procedures.14COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYMiSeq 提供多提供多种种reads数的试数的试剂盒剂盒支支持持不不同应同应用用MiSeqCore ConsumablesVersion 3600 cycles150 cyclesCore ConsumablesVersion 2 Micro300 cycles(Micro)1 Million reads4 Million reads15 Million reads25 Million readsCore Consumables
14、Version 2 Nano500 cycles(Nano)300 cycles(Nano)Core ConsumablesVersion 2500 cycles300 cycles150 cyclesFor Research Use Only.Not for use in diagnostic procedures.15COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYChemistryVersionNumber ofReads(mil)CyclesOutput(Gb)SequencingTime(hrs)32560015563251503.82121550
15、08.5392153005.124215500.855.524(Micro)3001.22821(Nano)5000.51921(Nano)3000.319MiSeq 提供多提供多种种reads数的试数的试剂盒剂盒支支持持不不同应同应用用For Research Use Only.Not for use in diagnostic procedures.16COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYTargetedResequencingIllumina-AmpliSeq PanelTruSight Panels TruSeq/Nextera Exom
16、eTruSeq/Nextera-xGen ExomeTruSeq Synthetic LongRead*RNA&Regulation Sample PrepTruSeq RNATruSeq Stranded mRNATruSeq Stranded Total RNATruSeq Small RNA TruSeq Targeted RNATruSeq ChIPDNA Sample PrepTruSeq PCR-Free TruSeq Nano NexteraNextera XT Nextera Mate PairIllumina提供完整的试提供完整的试剂盒剂盒制制备方备方案案17COMPANY
17、CONFIDENTIAL FOR INTERNAL USE ONLY18靶靶向测向测序序微微生物检生物检测测 Ampliseq for Illumina Trusight Panel系列 16s rRNA测序 微生物宏基因组测序COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYAmpliseq简单的建库简单的建库+Illumina自动化高质自动化高质量测量测序序集中支持集中支持订购简单故障处理及时可以选择在线下单操作快速操作快速1.5小时手动时间5-7小时全部时间Panel设设计计&下单流程简单下单流程简单使用AmpliSeq designer 设计功能强大而
18、且 简单需要选择的选项减少了，但是却可以达到更 好的设计效果为客户设计提供帮助For Research Use Only.Not for use in diagnostic procedures.19COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYAmpliSeq 产品组合产品组合For Research Use Only.Not for use in diagnostic procedures.8个现成产品30个团体产品3 Cellular&Molecular Research 3个细胞&分子生物学研究1Complex Disease Genomics 1
19、个复杂疾病基因组2Immunology 2个免疫相关2 Infectious disease 2个传染病相关1 Neuroscience1个神经性疾病相关5 Oncology 5个肿瘤相关16 Rare and Inherited Disease 16个罕见病&遗传病相关客户定制产品 DNA Pre-loaded genomes 2H-2018 Custom genomes Custom RNAComprehensive Cancer泛癌种Cancer Hotspot v2癌症热点 v2Focus肿瘤位点聚焦Comprehensive v3综合性肿瘤位点 v3BRCA乳腺癌Immune Res
20、ponse免疫应答Transcriptome Human Gex人类转录组基因研究Exome外显子组选项选项20COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY8个现成产品30个团体产品*可以定制96 amplicons-3000 rxns客户定制产品*可以定制96 amplicons-3000 rxns方便的小型包装 90%Coverage uniformity90%平均覆盖率 90%On target90%靶向率产品详情产品详情*Performance has not been validated by Illumina but observed per
21、formance is usually par with ready-to-use panels given typicaldesign and assay conditionsFor Research Use Only.Not for use in diagnostic procedures.AmpliSeq 产品组合产品组合1.5 小小时时 手动时间手动时间215 7小小时时 总时间总时间2.5天天 从从DNA到结果数据到结果数据22COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY8个现成产品个现成产品和和30个团个团体产体产品品Panel试剂盒试剂盒G
22、enes基因数基因数Application应用应用Input样本样本BRCA2BRCA1/2两个基因的外显子区域和侧翼内含子区，同时支持胚系和 体细胞突变检测DNACancer Hotspot v250癌基因中常发生突变的热点区域DNAFocus Panel52实体瘤相关的突变热点，包括SNV,indels,CNVs和基因融合DNA+RNAComprehensive v3161实体瘤相关的突变热点，包括SNV,indels,CNVs和基因融合，适合泛癌种检测和临床试验DNA+RNAComprehensive Cancer409覆盖409个癌基因的外显子区域，检测SNV和indelsDNAImm
23、une Response Panel395与免疫反应相关的395个基因的表达RNATranscriptome Human Gex20,800 RefSeq分析基因表达RNAExome57.7Mb人外显子组测序DNACommunity3个细胞&分子生物学研究；2个复杂疾病基因组；1个免疫相关；2个 传染病相关；1个神经性基本相关；5个肿瘤相关；16个罕见病和遗 传病相关DNA30个团体产品个团体产品Cardiac Arrhythmias and Cardiomyopathy Research Panel (心律不齐及心肌症研究试剂盒)(92 genes,2 pools,2,198 amplico
24、ns）Deafness Research Panel v2 (耳聋研究试剂盒,v2)(126 genes,2 pools,3,520amplicons)Autism Research Panel (孤独症研究试剂盒)(236 genes,2 pools,7,194amplicons)Endocrine Research Panel v2 (内分泌疾病研究试剂盒,v2)(340 genes,2 pools,6,359 amplicons)Cardiovascular Research Panel (心血管疾病研究试剂盒)(404 genes,2 pools,10,430 amplicons)De
25、rmatology Research Panel v2 (皮肤病研究试剂盒v2)(222 genes,2 pools,4,964amplicons)Dysmorphia-Dysplasia Research Panel v2(畸形-发育不良研究试剂盒)(519 genes,2 pools,12,558 amplicons)Gastrointestinal Research Panel v2(胃肠疾病研究试剂盒,v2)(194 genes,2 pools,4,103 amplicons)Hearing Loss Research Panel v1 (听力缺失研究试剂盒v1)(63 genes,2
26、 pools,2,064amplicons)Hematology Research Panel (血液疾病研究试剂盒)(394 genes,2 pools,7,434 amplicons)Inborn Errors of MetabolismResearch Panel v2(先天性代谢异常研究试剂盒,v2)(594 genes,2 pools,9,681amplicons)Noonan Research Panel (Noonan综合症研究试剂盒)(14 genes,2 pools,268 amplicons)Ophthalmic Research Panel (眼科疾病研究试剂盒)(316
27、 genes,2 pools)Primary Immune Deficiency Research Panel v2(原发性免疫缺陷研究试剂盒 v2)(264 genes,2 pools,5,241 amplicons)Pulmonary Research Panel v2 (肺部疾病研究试剂盒v2)(131 genes,2 pools,3,365amplicons)Renal Research Panel v2(肾脏疾病研究试剂盒v2)(154 genes,2 pools,4,332 amplicons)Epilepsy Research Panel (癫痫研究试剂盒)(385 genes,
28、2 pools,8,875amplicons)BRCA Plus,Extended Hereditary Breast and Ovarian Research Panel(BRCA及其它基因相关的遗传性 乳腺癌及卵巢癌研究试剂盒)(11 genes,3 pools,699 amplicons)Inherited Cancer Research Panel遗传性癌症研究试剂盒)(134 genes,2 pools,3,340 amplicons)Colon and Lung Research Panel v2(结直肠癌和肺癌研究试剂盒,v2)(22 genes,1 tube,92 amplic
29、ons)Long Non-Coding RNA Research Panel(癌症相关长链非编码RNA研究 试剂盒)(RNA,表达水平)438非编码RNA,1 pool,418 ampliconsRNA Fusion Lung Cancer Research Panel(肺癌相关RNA融合研究试剂盒)(RNA)(70 genes,1 pool,85 amplicons)Ebola Research Panel(埃博拉病毒研究试剂盒)(埃博拉病毒基因组,2 pools,150 amplicons)TB Research Panel(结核分支杆菌耐药性研究)(8 genes,1 pool,109
30、amplicons)Neurological Research Panel (神经疾病研究试剂盒)(751 genes,2 pools,18,077 amplicons）RNA Inflammation ResponseResearch Panel(免疫反应RNA表达水平研究试 剂盒)(RNA)(683 genes,1 pool,683amplicons)Dementia Research Gene Panel (痴呆基因研究试剂盒)(17 genes,2 pools,214amplicons)RNA MAPK Pathway ResearchPanel(MAPK信号通路RNA表达水平研 究试
31、剂盒)(RNA)(197 genes,1 pool,197amplicons)RNA Stem Cell Research Panel(干细胞RNA表达水平研究试剂 盒)(RNA)930 genes,1 pool,930 ampliconsRNA WNT Signaling PathwayResearch Panel(WNT信号通路RNA表达水平研 究试剂盒)(RNA)(169 genes,1 pool,169amplicons)传染病相关C神OM经PA性NY疾C O病N相FID关ENTIAL FOR INTERNAL USE ONLY罕见病和遗传病相关肿瘤相关免疫相关复杂疾病基因组研究相关细
32、胞分子生物学研究23COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY仪仪器产器产出出数数据据量量MiniSeqMiSeqiSeq 100Focus Panel(8 samples)BRCA Somatic(12 samples)Cancer Hotspot(16 samples)BRCA Germline(96 samples)24NextSeqImmune Response(24 samples)Focus Panel(48 samples)BRCA Somatic(80 samples)Cancer Hotspot(96 samples)BRCA Ger
33、mline(96 samples)Exome(8 samples)Comprehensive Cancer(12 samples)Transcriptome(40 samples)Comprehensive v3(48 samples)#扩增子数扩增子数1.Samples per run values assume recommended coverageAmpliseq简单的建库简单的建库+Illumina自动化高质自动化高质量测量测序序COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY轻松进行客轻松进行客户户定定制制DesignStudioFrom Dro
34、pdown,Select:Custom Assay Design with DesignStudioFrom Dropdown,Select:DesignStudioFor Research Use Only.Not for use in diagnostic procedures.25COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYDesignStudio 定制主页定制主页For Research Use Only.Not for use in diagnostic procedures.26COMPANY CONFIDENTIAL FOR INTERNA
35、L USE ONLY选选择适合择适合用用户的定户的定制制方方式式AmpliSeq GeneTargets include:1 Kb-5 Mb of ContentGenes,Exons,and/or Region CoordinatesAmpliSeq HotspotTargets include:1 Kb 5 Mb of Content Hotspot Regions and SNPsNextera Rapid CaptureTargets include:27 5 Mb of ContentLarge Gene Panels and/or Extended Exome ContentCOM
36、PANY CONFIDENTIAL FOR INTERNAL USE ONLYRNA-PanCancerInherited DiseaseHLACancerCardioTruSight 试剂盒组试剂盒组表现优秀的靶向测序试剂盒MyeloidTumor28COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY临床全临床全外外 Trusight One/Trusight One Expanded全面解读基因与疾病的关系Number of Genes4,813Target Region Size12 MbTargeted Exons62,00020 x Minimum
37、Coverage目标基因目标基因捕获捕获Number of Genes6700+Target Region Size16.5 MbTargeted Exons86,00020 x Minimum Coverage目标基因目标基因捕获捕获29COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY微微生物学生物学四四大主流大主流应应用用Sequence the entire genome and achieve accurate de novo assembly using cultures as starting material.Generate accurate
38、 reference genomes,for microbial identification30Explore all genes in all organisms present in many complex environmental or microbial samples,evaluate bacterial diversity and detect microbe abundance.Identify and compare bacteria present within a given sample.Clarify phylogenetic relationships betw
39、een bacteriaDetection of low-frequency viral particles within non-cultured samples.Identify,characterize and quantify DNA and RNA viral transcripts of infectious agents.全全基因基因组组测测序序鸟鸟枪法枪法宏宏基基因因组组测测序序16S rRNA测序测序病病毒毒学学COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYMiSeq DNA WorkflowPrepSequenceAnalyzeShar
40、eBaseSpaceNextera XT Nextera DNA FlexMiSeqMSR or BaseSpace15 minutes hands-on20 minutes hands-onFully automatedSecure and storeRapid library preparation Complete library prep in as little as 90 minutes with only 15 minutes of hands-on timeOptimized for small genomes,PCR amplicons and plasmids One li
41、brary prep kit for many applicationsUltra low input Only a single nanogram of input DNA neededInnovative sample normalizationEliminates the need for library quantification before sample pooling and sequencingFor Research Use Only.Not for use in diagnostic procedures.31COMPANY CONFIDENTIAL FOR INTERN
42、AL USE ONLYOrder amplicon primersPCR amplify V3 and V4 regions of 16S rRNAgeneAdd dual-index barcodes by PCRLibrary QC and NormalizationLibrary Pooling3216S 宏基因组测宏基因组测序序流流程程Illumina提供标准化流程COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY人类健康人类健康遗传病遗传病感染性感染性疾疾病病科学研究科学研究生殖生殖健健康康法医学法医学健康筛查健康筛查肿瘤肿瘤生物制药生物制药农业农业illumina 高通量检测高通量检测覆盖覆盖各各个个领领域域33COMPANY CONFIDENTIAL FOR INTERNAL USE ONLYThank you Questions?34
