Welcome to. Basic Analysis of NanoString Gene Expression Data: QC, Normalization, and Differential Expression Testing
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1 Welcome to Basic Analysis of NanoString Gene Expression Data: QC, Normalization, and Differential Expression Testing Today s Presenter: Ryan S. Friese, Ph.D, Field Applications Scientist for Southern California Today s Moderator: Erin K. Brown, Associate Technical Services Scientist, Seattle This Webinar will be recorded and made available to you Attendees will be hidden from each other and muted throughout the presentation For questions, please use the Chat feature to ask your question to the Moderator If your question will be answered in the course of the presentation, the Moderator will let you know. If your question is pertinent at the time, the Moderator will either communicate to the presenter to address it to the group or answer you directly via chat. The Presenter will open the floor for questions at the end. Thank you for attending 1 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
2 Agenda 1. Technology overview 2. Data import 3. Data QC 4. Normalization 5. Differential Expression 6. Visualizations Heat maps Scatter plots Box plots 2 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
3 The Only Direct, Digital Counting Technology Novel chemistry invented in Leroy Hood s lab at the Institute for Systems Biology Digital counting technology Probes up to 800 targets simultaneously Single-molecule, fluorescent barcodes, each attached to an individual nucleic acid molecule 3 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
4 Digital Counting using Barcoded Probes Half Site 50 bases Half Site 50 bases Barcode Biotin Target-specific Capture Probe Target-specific Reporter Probe Barcode Identity XLSA FOX5 PDCD1 4 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
5 Enzyme-free Digital Counting Directly Hybridize Lysate or Total RNA Only manual process Only 4 pipetting steps per sample required No amplification or reverse transcription 800 hybridizations in a single tube (Amplification may be needed for single cell application for low concentrations) 5 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
6 Digital Counting using Barcoded Probes 6 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
7 Digital AND Automated: Reproducible and Precise Automated instrumentation excess probes removed Purification Probe binding to slide Imaging 7 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
8 Log 2 Digital Data: One barcode = one mrna molecule Gene Sample 1 Sample 2 Sample 3 SPP1 8, GAPDH 7,452 1,621 1,370 PLA2G2A 6, Fold change relative to sample 1 PDCD1 2, TGFBI 2, , TIMP1 2, PGK1 1,427 1, MCL1 1,320 1, FAT1 1, STAT3 1,270 1,554 1,054 Slide image PLG 1,129 7, XRCC5 1,113 1,854 1,791 COL1A1 1, , Sample 2 Sample 3 ERBB2 1, FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
9 Quality Control Metrics in nsolver Software 9 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
10 Imaging QC Binding Surface 555 Field of View (FOV) on slide surface area Imaging QC = (FOV Counted) / (FOV Count) 10 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
11 Binding Density QC Binding Density = Number of fluorescent spots per square micron High density indicates that barcodes could be too close to each other for the imaging to successfully resolve them. Upper limit of binding density is conservative. A QC flag doesn t necessarily mean assay failure! 11 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
12 Positive and Negative Controls Internal controls based on ERCC sequences. Spiked-in to every sample. Contained in probe reagent tubes. Positive controls In vitro transcribed RNA spike-ins Linear dilution series, six concentrations 0.125, 0.5, 2, 8, 32,128 fm Negative controls No target RNA is spiked in Used to establish background or limit of detection (LOD) 12 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
13 Log2 (raw counts) Positive Control Linearity QC Linearity of Positive Controls R² = POS_B POS_A POS Control Concentration (fm) Raw Counts POS_A POS_B POS_C POS_D POS_E POS_F POS_E POS_D POS_C Note: As POS_F is considered to be below limit of detection, POS_F is not included in the linearity check Log2 (concentration, fm) POS Control Linearity QC: R 2 of concentration vs raw counts 13 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
14 Log2 (raw counts) Limit of Detection QC Linearity of Positive Controls R² = POS_B POS_A POS Control Concentration (fm) Raw Counts POS_A POS_B POS_C POS_D POS_E POS_F POS_E POS_C POS_D Average NEG + 2*SD Log2 (concentration, fm) NEG Control Raw Counts NEG_A 21 NEG_B 11 NEG_C 5 NEG_D 5 NEG_E 15 NEG_F 19 NEG_G 8 NEG_H 3 Limit of Detection QC: POS_E > Average of NEG controls + 2*SD 14 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
15 NanoString nsolver Webinar Series For additional support, please contact NanoString Support at: (888) Join us for our next nsolver webinars: nsolver Basics webinar in August. nsolver Advanced Analysis in September. Slides, sample data, and a recording will be made available after the webinar is complete. Thank you for attending! 15 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
16 Thank You 16 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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