MiSeq高通量测序在临床的应用介绍课件.pptx

MiSeq高通量测序在临床的应用介绍,Illumina,MiSeqIllumina,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,Library Preparation,Data Analysis,Cluster Generation,Sequencing,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,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 Libraryshown,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,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,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,簇生成过程,接头序列,3 extension3 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DNA变性原始模板链,Discard 洗去丢弃,单链DNA,桥式PCR扩增,双链DNA桥式结构变性,桥式PCR扩增,5,COMPANY CONFIDENTIAL FOR INT,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,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,100 Microns,7,ClustersDNA簇,COMPANY CONFIDENTIAL FOR INT,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,9,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,i5 Index Seq Primer,(SBS3rev/BP13),i5 Index,Index Read 2 (i5),i7 Index,1,2,3,Pair,ed End around,Turn,4,1,2,3,Paired-End Turnaround,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,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 EnrichmentProcesses WGS reads using BWA for alignmentUses GATK for variant detectionEnrichmentPerforms read mapping using IsaacOffers 4x faster alignment with same accuracyas 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 | 25M2x300,MiSeq,7.5 Gb | 25M2x150,MiniSeq,120 Gb | 400M2x150,NextSeq550,167 6000 Gb | 1.6 20B2x150,Mid, High,Micro, Nano V2, V3,Mid High,S1,S2,S4NovaSeq 6000,桌面式测序仪,HiSeq X,1300-1800Gb|5.3-6B2x150,生产级测序仪,1.2 Gb | 4M 2x150,11,iSeq,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 panelsAmplicon DNA and RNA available,小基因组测序Sequence small genomes with ease,辅助性应用Off-the-shelf, optimized solutionsIntuitive workflows for fast implementation,2x150base pairs,1.2gigabases,4Mreads,917hour run time,12COMPANY CONFIDENTIAL FOR I,COMPANY CON13,FIDENTIAL FOR INTERNAL USE ONLY,MiSeq 的优点,For Research Use Only. Not for use in diagnostic procedures.,COMPANY CONFIDENTIAL FOR INT,COMPAN,Y CONFIDENTIAL FOR INTERNAL USE ONLY,MiSeq System,PE300 | 75% Q300.3 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,MiSeq,Core ConsumablesVersion 3,600 cycles150 cycles,Core ConsumablesVersion 2 Micro,300 cycles (Micro),1 Million reads,4 Million reads15 Million reads,25 Million reads,Core ConsumablesVersion 2 Nano,500 cycles (Nano)300 cycles (Nano),Core ConsumablesVersion 2,500 cycles300 cycles150 cycles,For Research Use Only. Not for use in diagnostic procedures.,15,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,For Research Use Only. Not for use in diagnostic procedures.,16,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,TargetedResequencing,Illumina-AmpliSeq PanelTruSight Panels TruSeq/Nextera ExomeTruSeq/Nextera-xGen ExomeTruSeq Synthetic LongRead*,RNA & Regulation Sample Prep,TruSeq RNATruSeq Stranded mRNATruSeq Stranded Total RNATruSeq Small RNA TruSeq Targeted RNATruSeq ChIP,DNA Sample Prep,TruSeq PCR-Free TruSeq Nano NexteraNextera XT Nextera Mate Pair,Illumina提供完整的试剂盒制备方案,17,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,18,靶向测序,微生物检测, Ampliseq for Illumina Trusight Panel系列, 16s rRNA测序 微生物宏基因组测序,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,Ampliseq简单的建库+Illumina自动化高质量测序,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1.5小时手动时间5-7小时全部时间Panel设计&下单流程简单使用AmpliSeq designer 设计功能强大而且 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.,19,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,AmpliSeq 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Complex Disease Genomics 1个复杂疾病基因组Immunology 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,DNAPre-loaded genomes2H-2018,Custom genomesCustom RNA,Comprehensive 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 Response免疫应答Transcriptome Human Gex人类转录组基因研究Exome外显子组,选项,20,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,8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,*Performance has not been validated by Illumina but observed performance 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,1.5 小时 手动时间,21,5 7小时 总时间,2.5天 从DNA到结果数据,COMPANY CONFIDENTIAL FOR INT,22,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,8个现成产品和30个团体产品,22COMPANY CONFIDENTIAL FOR I,30个团体产品,传染病相关,C神OM经PA性NY疾CO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胞分子生物学研究,23,30个团体产品Inborn Errors of Metabo,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,仪器产出数据量,MiniSeq,MiSeq,iSeq 100,Focus Panel (8 samples)BRCA Somatic (12 samples)Cancer Hotspot (16 samples)BRCA Germline (96 samples),24,NextSeqImmune Response (24 samples)Focus Panel (48 samples)BRCA Somatic (80 samples)Cancer Hotspot (96 samples)BRCA Germline (96 samples),Exome (8 samples)Comprehensive Cancer (12 samples)Transcriptome (40 samples)Comprehensive v3 (48 samples),# 扩增子数,1. Samples per run values assume recommended coverage,Ampliseq简单的建库+Illumina自动化高质量测序,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,轻松进行客户定制DesignStudio,From Dropdown, Select:Custom Assay Design with DesignStudio,From Dropdown, Select: DesignStudio,For Research Use Only. Not for use in diagnostic procedures.,25,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,DesignStudio 定制主页,For Research Use Only. Not for use in diagnostic procedures.,26,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,选择适合用户的定制方式,AmpliSeq GeneTargets include:1 Kb - 5 Mb of ContentGenes, Exons, and/or Region Coordinates,AmpliSeq HotspotTargets include:,1 Kb 5 Mb of Content Hotspot Regions and SNPs,Nextera Rapid CaptureTargets include:,27,5 Mb of ContentLarge Gene Panels and/or Extended Exome Content,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,RNA-PanCancer,Inherited Disease,HLA,Cancer,Cardio,TruSight试剂盒组表现优秀的靶向测序试剂盒,MyeloidTumor,28,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,临床全外 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 Coverage,目标基因捕获,Number of Genes6700+Target Region Size16.5 MbTargeted Exons86,00020 x Minimum Coverage,目标基因捕获,29,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,微生物学四大主流应用,Sequence the entire genome and achieve accurate de novo assembly using cultures as starting material. Generate accurate reference genomes, for microbial identification,30,Explore 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 between bacteria,Detection of low-frequency viral particles within non- cultured samples. Identify, characterize and quantify DNA and RNA viral transcripts of infectious agents.,全基因组测序,鸟枪法宏基因组测序,16S rRNA测序,病毒学,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,MiSeq DNA Workflow,Prep,Sequence,Analyze,Share,BaseSpace,Nextera XT Nextera DNA Flex,MiSeq,MSR or BaseSpace,15 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 library 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.,31,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,Order amplicon primers,PCR amplify V3 and V4 regions of 16S rRNAgene,Add dual- index barcodes by PCR,Library QC and Normalization,Library Pooling,32,16S 宏基因组测序流程Illumina提供标准化流程,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,人类健康,遗传病,感染性疾病,科学研究,生殖健康,法医学,健康筛查,肿瘤,生物制药,农业,illumina高通量检测覆盖各个领域,33,COMPANY CONFIDENTIAL FOR INT,COMPANY CONFIDENTIAL FOR INTERNAL USE ONLY,Thank you Questions?,34,COMPANY CONFIDENTIAL FOR INT,