Affymetrix åºå 表ç¾æ¶ç åæè»é«ä»ç´¹
Affymetrix 基因表現晶片 分析軟體介紹 顏吟紋 冷泉港生物科技股份有限公司 Agenda Expression GeneChip Introduction Analysis Workflow Expression Console Demo TAC 3.0 Demo Affymetrix Confidential 1 For research use only. Not for use in diagnostic procedures. Affymetrix 基因表現晶片 簡介 Affymetrix Array Types Isoform 1 Isoform 2 5' 3' Affymetrix 3' IVT Competitors (Illumina, Agilent) Affymetrix WT Exon Affymetrix WT Gene HTAv2 Human Transcriptome Array Affymetrix Confidential 3 For research use only. Not for use in diagnostic procedures. Array Type Comparison Array Type U133 plus 2 Human Gene 2.0 HTA 2.0 3’-IVT Whole Transcript Whole Transcript Genes >38,500 >30,000 44,699 Transcripts 47,400 Splice junction (probe set) - - 339,146 Genes - >11,000 22,829 Transcripts - Protein-coding 245,349 Non-protein coding Affymetrix Confidential 40,914 4 For research use only. Not for use in diagnostic procedures. Evolution of Human Whole Transcript Arrays Year 2004 Array Status Target Strand Exon 1.0 ST Commercialized Sense Gene 1.0 ST Commercialized Sense 2006 HJAY Array Tech Access Sense HTA 1.0 (AKA GGH) Tech Access AntiSense 2012 Gene 2.0 ST Commercialized Sense 2013 HTA 2.0 Array Commercialized Sense Affymetrix Confidential 5 Probe Coverage • 4 per exon • ~28 per gene • 8 per exon • 8 per junction • 10 per exon • ~4 per junction • ~26 per gene • 10 per exon • 4 per junction. Content • well annotated • speculative • well annotated • well annotated • well annotated • well annotated • well annotated For research use only. Not for use in diagnostic procedures. Affymetrix 涵蓋物種 人類 小鼠 大鼠 阿拉伯芥 大麥 牛 犬 線蟲 雞 柑橘 棉花 果蠅 大腸桿菌 玉米 斑馬魚 Affymetrix Confidential 苜蓿 綠膿桿菌 瘧原蟲/瘧蚊 白楊木 豬 恆河猴 稻米 金黃色葡萄球菌 大豆 甘蔗 釀酒葡萄 小麥 非洲爪蟾 兔子 酵母 6 For research use only. Not for use in diagnostic procedures. We Have More Than Just Mature miRNA Nearly all small non-coding RNA! scaRNA Small Cajal Body RNA Modifies RNA Polymerase snoRNA Mature miRNA Regulates translation of mRNA into proteins Affymetrix Confidential Small Nucleolar RNA Guides rRNA modifications Pre-miRNA Precursors to mature miRNA 7 For research use only. Not for use in diagnostic procedures. miRNA Array contents miRNA 2.0 miRNA 3.0 miRNA 4.0 miRBase release (version) 15 17 20 Organism 131 153 203 Total Probe Sets 15,644 19,724 30,424 Human mature miRNA probe sets 1,105 1,733 2,578 Mouse mature miRNA probe sets 722 1,111 1,255 Rat mature miRNA probe sets 389 680 728 Human snoRNA and scaRNA probe sets 2,334 2,216 1,996 Human pre-miRNA probe sets 1,105 1,658 2,025 Mouse pre-miRNA probe sets 690 855 1255 Rat pre-miRNA probe sets 389 486 490 Affymetrix Confidential 8 For research use only. Not for use in diagnostic procedures. New Affymetrix Expression Analysis Software Updated Expression Console and New Transcriptome Analysis Console for the Detection of Differentially Expressed Genes and Alternative Splicing Events CEL file→CHP file Affymetrix Confidential CHP file→Condition analysis 9 For research use only. Not for use in diagnostic procedures. Analysis Workflow Import CEL File Perform gene—level normalization and signal summarization Perform exon—level normalization and signal summarization Select analysis: 1. Gene-level 2. Exon-level 3. Alternative splicing Expression Console (EC1.4) Software - Normalization & QC Transcriptome Analysis Console (TAC3.0) Software - Result visualization 1. Import CHP File 2. Create condition 3. Run analysis Gene results visualization Affymetrix Confidential Exon results visualization Splicing results visualization 10 For research use only. Not for use in diagnostic procedures. Expression Console Demo Analysis Workflow Import CEL File Perform gene—level normalization and signal summarization Perform exon—level normalization and signal summarization Select analysis: 1. Gene-level 2. Exon-level 3. Alternative splicing Expression Console (EC1.4) Software - Normalization & QC Transcriptome Analysis Console (TAC3.0) Software - Result visualization 1. Import CHP File 2. Create condition 3. Run analysis Gene results visualization Affymetrix Confidential Exon results visualization Splicing results visualization 12 For research use only. Not for use in diagnostic procedures. System Recommended Requirement Affymetrix Confidential 13 For research use only. Not for use in diagnostic procedures. EC 1.4 Installation 1. Go to www.affymetrix.com and navigate to the following location: Home > Products > Microarray Solutions > Instruments and Software > Software > Expression Console 2. Locate and download the zipped EC1.4 software package. 3. Unzip the file, then double-click ECx64.exe to install it. 4. Follow the directions provided by the installer. Affymetrix Confidential 14 For research use only. Not for use in diagnostic procedures. Library & Annotation Installation 2 3 1 Affymetrix Confidential 15 For research use only. Not for use in diagnostic procedures. Creating a Study 1 2 Add CEL file Affymetrix Confidential 16 For research use only. Not for use in diagnostic procedures. Editing Sample Attributes Select File →Open Sample/Array Attribute File and choose one or more sample files(ARR) Affymetrix Confidential 17 For research use only. Not for use in diagnostic procedures. Viewing QC Sample Metrics: To identify the outlier samples A sample should be flagged and possibly removed when several metrics are outliers within the distribution of samples. - Pos_vs_neg_auc (The value should be above 0.8) - All probe Set Mean - All probe Set RLE Mean Affymetrix Confidential 18 For research use only. Not for use in diagnostic procedures. Viewing QC Hybridization Metrics - hybridization control: BioB<BioC<BioD<Cre Labeling Metrics - labeling control: Lys<Phe<Thr<Dap Affymetrix Confidential 19 For research use only. Not for use in diagnostic procedures. PCA graph Help identify outliers using PCA algorithm Click point(s) in PCA graph to highlight sample(s) in Study Window Samples can be labeled by color and shape with user attributes (ARR file) By Strain By Growth Condition GeneChip Arabidopsis ATH1 Genome Array Affymetrix Confidential 20 For research use only. Not for use in diagnostic procedures. Exporting Data Export Tables and Graphs to PDF Export Probe Set Report with Annotation to TXT (To create annotation merge file) Affymetrix Confidential 21 For research use only. Not for use in diagnostic procedures. TAC 3.0 Demo Analysis Workflow Import CEL File Perform gene—level normalization and signal summarization Perform exon—level normalization and signal summarization Select analysis: 1. Gene-level 2. Exon-level 3. Alternative splicing Expression Console (EC1.4) Software - Normalization & QC Transcriptome Analysis Console (TAC3.0) Software - Result visualization 1. Import CHP File 2. Create condition 3. Run analysis Gene results visualization Affymetrix Confidential Exon results visualization Splicing results visualization 23 For research use only. Not for use in diagnostic procedures. System Minimum Requirement Microsoft Windows® XP with Service Pack 2.0 Operating system Processor Memory (RAM) Available disk space Web browser 64-bit 2.4 GHz Intel® Pentium® Quad Core 8 GB, (recommended 16 GB) 150 GB HD + data storage Internet Explorer® 7.0 and above Microsoft Windows® 7 Professional with Service Pack 1 64-bit Operating system Processor Memory (RAM) Web browser Affymetrix Confidential 2.8 GHz Intel® Pentium® Quad Core 8 GB, (recommended 16 GB) Internet Explorer® 7.0 and above 24 For research use only. Not for use in diagnostic procedures. TAC 3.0 Installation 1. Go to www.affymetrix.com and navigate to the following location: Home > Products > Microarray Solutions > Instruments and Software > Software > TAC 2. Locate and download the zipped TAC 3.0 software package. 3. Unzip the file, then double-click TAC3x64.exe to install it. 4. Follow the directions provided by the installer. Affymetrix Confidential 25 For research use only. Not for use in diagnostic procedures. Library Installation 1. 2. Affymetrix Confidential 26 For research use only. Not for use in diagnostic procedures. Set up a analysis 1. 2. 3. 4. 5. At the main TAC window, click one analysis button. Import CHP file data in the sample file window. Import CHP file into condition groups. Choose ANOVA setting, paired or unpaired. Click “Run Analysis” button Array Type HTA 2.0, MTA 1.0, RTA 1.0 All expression array, miRNA Exon array, Gene array Affymetrix Confidential 27 For research use only. Not for use in diagnostic procedures. Result visualization Summary Table Affymetrix Confidential 28 For research use only. Not for use in diagnostic procedures. Table Options Rearranging Columns Showing or Hiding Table Columns Sorting Columns Filtering Column Data Copying Column Data Changing Comparison Searching Key Words Customizing Table Columns Exporting Report Copying Selected ID, Rows, and Sequence Accessing External Databases Affymetrix Confidential 29 For research use only. Not for use in diagnostic procedures. Graph Types Scatter Plot Graph Volcano plot Graph Chromosome Summary Graph Hierarchical Clustering Graph Signal Bar Graph (in all condition comparison) Affymetrix Confidential 30 For research use only. Not for use in diagnostic procedures. Dynamic Visualization Tables and Plots Lasso interesting groups and see in the table. Affymetrix Confidential 31 For research use only. Not for use in diagnostic procedures. Additional Plots Affymetrix Confidential 32 For research use only. Not for use in diagnostic procedures. miRNA-mRNA Network Interaction • Click “View Interaction Network” to set up miRNAmRNA network. Affymetrix Confidential 33 For research use only. Not for use in diagnostic procedures. New Feature of TAC 3.0-Pathway Affymetrix Confidential 34 For research use only. Not for use in diagnostic procedures. Relevance of Alternative Splicing One gene can have two transcripts with opposite effects Bcl-x Bcl-xS Bcl-xL Inhibits apoptosis Activates apoptosis Cell 1993 Boise et al., Aug 27;74(4):597-608 Affymetrix Confidential 35 For research use only. Not for use in diagnostic procedures. Splicing Analysis - Only for HTA 2.0, MTA1.0, and RTA 1.0 - This method is not for de novo event discovery – only events with isoform evidence will be called. Affymetrix Confidential 36 For research use only. Not for use in diagnostic procedures. Five Classic Modes of Alternative Splicing CE: One exon is spliced out of the primary transcript together with its flanking introns. (exon 1-2-3 or 1-3) Exon skipping MXE: Two consecutive exons that are never both included in the mature mRNA transcript (exon 1-2-4 or 1-3-4) Mutually exculsive exons Alternative 5’ donor sites Alternative 3’ acceptor sites Intron retension Affymetrix Confidential Alt 5SS: Two or more splice sites are recognized at the 3' end of an exon. An alternative 5' splice junction (donor site) is used, changing the 3' boundary of the upstream exon. (exon 1L-2 or 1S-2) Alt 3SS: Two or more splice sites are recognized at the 5' end of an exon. An alternative 3' splice junction (acceptor site) is used, changing the 5' boundary of the downstream exon (exon 1-2L or 1-2S) IR: A sequence is spliced out as an intron or remains in the mature mRNA transcript 37 For research use only. Not for use in diagnostic procedures. Frequently Used Terms 1) Probe Selection Region (PSR) 1.1) 1 PSR = 1 Exon 1.2) n PSR = 1 Exon Affymetrix Confidential 38 For research use only. Not for use in diagnostic procedures. Frequently Used Terms 2) Spacer A Spacer represents a transcript cluster (TC) probe selection region (PSR) where a selection of probes is not possible. Spacer is typically a region with less than 25 bases. Occasionally, some of them can exceed 25 bases Spacer doesn’t have data Affymetrix Confidential 39 For research use only. Not for use in diagnostic procedures. Frequently Used Terms 3) Inclusion and Exclusion Junctions 3.1) Inclusion Junction: detects 2 neighboring PSRs. 3.2) Exclusion Junction: detects PSRs that are not genomic neighbors. Affymetrix Confidential 40 For research use only. Not for use in diagnostic procedures. Frequently Used Terms 4) Splicing Index 40 4 2 2 1 1 10 +: condition 1 > condition 2 - : condition 1< condition 2 Affymetrix Confidential 41 For research use only. Not for use in diagnostic procedures. Splicing Event Estimate - CE Pattern • For CE, we are looking for a pattern that PSR and its two inclusion junctions are changing in the same direction, but exclusion junction is changing in the opposite direction. • PSR’s direction decides junctions’ directions Condition 1 Affymetrix Confidential Condition 2 42 For research use only. Not for use in diagnostic procedures. Splicing Event Estimate - MXE Pattern • For MXE, we are looking for a pattern that one set of PSR and its inclusion and exclusion junctions are changing in the opposite direction of another set. • PSR’s direction decides junctions’ directions Condition 2 Condition 1 Affymetrix Confidential 43 For research use only. Not for use in diagnostic procedures. Splicing Event Estimate – Alt 5SS Pattern • For Alt 5SS, we are looking for a pattern that PSR and its inclusion junction are changing in the same direction, but exclusion junction is changing in the opposite direction • PSRX and PSR1 shall be different and they are genomic neighbors • PSR’s direction decides junctions’ directions; PSR1’s direction decides PSRX’s direction Condition 1 Affymetrix Confidential Condition 2 44 For research use only. Not for use in diagnostic procedures. Splicing Event Estimate – Alt 3SS Pattern • For Alt 3SS, we are looking for a pattern that PSR and its inclusion junction are changing in the same direction, but exclusion junction is changing in the opposite direction • PSRX and PSR1 shall be different and they are genomic neighbors • PSR’s direction decides junctions’ directions; PSR1’s direction decides PSRX’s direction Condition 1 Affymetrix Confidential Condition 2 45 For research use only. Not for use in diagnostic procedures. Splicing Event Estimate – IR Pattern • For IR, we are looking for a pattern that a PSR is changing compare to its neighboring PSRX and PSRY • They must be genomic neighbors Condition 1 Affymetrix Confidential Condition 2 46 For research use only. Not for use in diagnostic procedures. How We Estimate Splicing Event – Event Score Event score - how well data fits the pre-defined splicing event patterns - Event score: 0 to 1, 1 is the highest possible score PSRs and its related junctions all contribute to event score - PSRs and Junctions can contribute positively or negatively to the event score depends on whether it goes to the required direction or not (defined by the patterns mentioned earlier). - Only PSRs get event score Affymetrix Confidential 47 For research use only. Not for use in diagnostic procedures. Example Event Score for PSR 1 for CE Event CE model Affymetrix Confidential 48 For research use only. Not for use in diagnostic procedures. Other tools • A software provider for microarray analysis. • Provide the complete analysis for the Biologist. • Validate your microarray data • Single tube multiplexing - up to 800 genes or regions, no amplification* required Affymetrix Confidential 49 For research use only. Not for use in diagnostic procedures. Thank you 顏吟紋 冷泉港生物科技股份有限公司 E-mail: [email protected]
© Copyright 2025