WHAT WOULD TREX DO? From Experimental Design to Analysis, the TREX - - PowerPoint PPT Presentation

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WHAT WOULD TREX DO? From Experimental Design to Analysis, the TREX - - PowerPoint PPT Presentation

Mar 23, 2024 466 likes •864 views

Your Research Question WHAT WOULD TREX DO? From Experimental Design to Analysis, the TREX Approach EXPERIMENTAL DESIGN What are my research goals? FFPE Clinical Where are my samples coming from? Fresh Tissue/Cells How much RNA

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WHAT WOULD TREX DO?

From Experimental Design to Analysis, the TREX Approach

Your Research Question

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EXPERIMENTAL DESIGN

  • What are my research goals?
  • Where are my samples coming from?
  • How much RNA will I have?
  • What is the expected quality of that RNA?
  • How many replicates to I need?
  • What is the data analysis going to look like?

FFPE Clinical Fresh Tissue/Cells FACS Sorted Cells

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GENE EXPRESSION ANALYSIS

Control Treatment Gene Expression Variation Gene Expression Variation

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EXPERIMENTAL DESIGN

  • What are my research goals?
  • Where are my samples coming from?
  • How much RNA will I have?
  • What is the expected quality of that RNA?
  • How many replicates to I need?
  • What is the data analysis going to look like?
  • How many samples do I need?
  • How much money do I have?

Clinical Cultured Cells Biological > Technical

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SAMPLE PREPARATION/ EXTRACTION

  • How did you extract your RNA?
  • What does the RNA QC look like?
  • Nanodrop: looks at chemical impurities and

quantity

  • Fragment Analyzer: Looks at RNA integrity
  • Qubit: quantity of material
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NANODROP

  • Spectrophotometer
  • Quantity > 20ng/uL
  • Contaminants
  • Salts
  • Proteins
  • Phenolics
  • Carbohydrates/Sugars
  • 260/230 Ratio: ~2-2.2
  • 260/280 Ratio: ~1.8-2

260/230 = 0.81 260/280 = 1.82 260/230 = 2.09 260/280 = 1.65

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FRAGMENT ANALYZER

  • Measure of RNA Integrity: RQN (RNA Quality

Number

  • Detect DNA contamination
  • Comparable Instruments
  • Tapestation
  • Bioanalyzer
  • NOT for quantitative analysis

Lower Marker Small RNA Small Ribosomal Subunit Large Ribosomal Subunit RQN:9.6 RQN:6.8 RQN:2.3 DNA Contamination

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QUBIT FLUOROMETER

  • RNA Quantity < 20
  • Can also detect DNA

contamination

  • Free to use in Genomics

Core

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RNASEQ LIBRARY PREP

NEB Next Ultra II RNASeq

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ILLUMINA SEQUENCING

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ILLUMINA SEQUENCING

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ILLUMINA SEQUENCING

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SEQUENCING

  • What Read Length do you want?
  • Our standard is 75bp
  • smRNA ideal is 50bp
  • SNP detection: Paired End
  • Transcriptome Assembly Longest read possible
  • How many reads do you need?
  • Our standard: 20m reads
  • smRNA standard: 10m reads
  • Isoform detection: much higher
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Data QC

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MULTIQC SUMMARY REPORT

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RQN 6.8

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Diagnostic Plots

  • BioAnalyzer Trace;
  • MultiQC Report (Alignment Statistics);
  • GeneBody Coverage;
  • Principal Components Analysis;
  • Hierarchical Clustering;

Overall quality Biological Signal

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Diagnostic Plots

  • BioAnalyzer Trace;
  • GeneBody Coverage;
  • Principal Components Analysis;
  • Hierarchical Clustering;
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  • GeneBody Coverage;
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Diagnostic Plots

  • BioAnalyzer Trace;
  • GeneBody Coverage;
  • Principal Components Analysis;
  • Hierarchical Clustering;
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  • Principal Components Analysis;

Color = Treatment Group Multiple data points with same color indicate biological reps

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Diagnostic Plots

  • BioAnalyzer Trace;
  • GeneBody Coverage;
  • Principal Components Analysis;
  • Hierarchical Clustering;
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  • Hierarchical Clustering;

Bottom-up approach

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RQN 6.8

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RQN 6.8 Borderline OKAY sample

Case Study 1

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RQN 7.1

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RQN 7.1

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Case Study 2

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Summary Report

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TREX Analysis Reports

  • MultiQC Alignment Summary;
  • Data QC Report;
  • Raw Count Table;
  • DE Genes Analysis Excel File;
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Next Steps…

  • DE Assessment of candidate genes;
  • DE genes (Panther/DAVID); GO-Term and Pathway

Enrichment;

  • Gene Set Enrichment Analysis (Broad);
  • Ingenuity Pathway Analysis (IPA);
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SLIDE 39

Thank You for Listening !

Contact Info: http://rnaseqcore.vet.cornell.edu/ E-mail List Serve: TREX-GENEREG-L Jen Grenier: jgrenier@cornell.edu Christine Butler: cab18@cornell.edu Ann Tate: aef93@cornell.edu Faraz Ahmed: fahmed@cornell.edu