ncounter TM Analysis System

ncounter TM Analysis System Molecules That Count TM www.nanostring.com

Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibilty ncounter Applications to Cancer Analysis

Key Milestones in NanoString History 2 23 26 28 29

Discovery to Dx Discovery Validation Routine testing Whole Genome Gene Expression NanoString NanoString 1-1 s samples 1, s genes 1-1 s samples 1 s 5 genes 1, s -1, s samples 1 s 5 genes

Gene Expression Technology Fit NanoString enables sensitive analysis of hundreds of genes QPCR NanoString Sensiti ivity Microarrays Multiplexing Capability (# of Transcripts)

ncounter Analysis System Up to 576 genes per rxn Digital Detection and Analysis No Enzymes Required Easy-to-use System Fully Automated Highly Sensitive, Reproducible Data

ncounter Analysis System ncounter Prep Station Fully automated sample processing Processes 12 samples in 2 hrs Uses pre-packaged consumables ncounter Digital Analyzer Fully automated data collection Up to 6 cartridges per run 24 hour processing unattended Over 41, data points in 24 hrs

Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

Probe Architecture Biotin Target Specific Capture Probe Target Specific Reporter Probe Target Specific Capture & Reporter Probes are designed that bind to the mrna transcript

Probe Architecture Target Specific Capture Probe Target Specific Reporter Probe Target Specific Capture & Reporter Probes are designed that bind to the mrna transcript

ncounter CodeSet 6 5 4 3 2 1 2 4 6 8 1 12 Target Specific Capture Probe System Controls Target Specific Reporter Probe Sequence Specific Capture Probes, Reporter Probes and System Controls are Combined to Create Your Custom CodeSet

Comprehensive Internal Controls Hybridization controls Positive controls are synthetic RNA targets spiked in at known concentrations (.1fM 1fM) Ensure proper hybridization conditions and no RNase contamination Used as a titration curve to quantitate endogenous transcripts Limit of quantification threshold Negative controls are reporter probe pairs for which no known target should exist within the endogenous sample Used when performing a Student s T-Test to determine presence or absence of an endogenous transcript Purification controls Positive & negative Alignment and immobilization controls

ncounter Assay Automated Process Hybridize CodeSet to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes mrna Capture & Reporter Probes

ncounter Assay Automated Process ncounter Prep Station Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Hybridized mrna Excess Reporters

ncounter Assay Automated Process ncounter Prep Station Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Surface of cartridge is coated with streptavidin Hybridized Probes Bind to Cartridge

ncounter Assay Automated Process ncounter Prep Station Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Reporters are aligned and immobilized for imaging and barcode counting

ncounter Assay Automated Process ncounter Digital Analyzer Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Image Surface One reporter code = 1 mrna

ncounter Assay Automated Process ncounter Digital Analyzer Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Code Gene Count x 3 y 1 z 2 Codes are counted and tabulated

Data Output Simple enough to analyze in Excel TM

Simple Protocol Buffer & CodeSet Sample Overnight Solution Phase Hybridization Automated Processing: 12 samples in 2 hr Collect Data: 2 min/sample

Validation Study Design Example 35 3 Reaction Number Comparison Calculator Number of genes 1 Number of samples 1 Technical Replicates 3 NanoString qpcr 3 3, No. rxns to complete study 25 2 15 1 5 NanoString QPCR Validate all the genes of interest to identify the most informative set Interrogate larger sample populations Reduce time to answer Reduce Complexity Without Reducing Content

Validation Study Design Example 35 3 Reaction Number Comparison Calculator Number of genes 2 Number of samples 1 Technical Replicates 3 NanoString qpcr 3 6, No. rxns to complete study 25 2 15 1 5 NanoString QPCR Validate all the genes of interest to identify the most informative set Interrogate larger sample populations Reduce time to answer Reduce Complexity Without Reducing Content

Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

Assay Reproducibility Replicate e 1 Counts Reproducibility of NanoString Assay Technical Replicates 1 1 1 1 1 R 2 =.9999 1 1 1 1 1 1 1 Replicate 2 Counts Data Courtesy of Dr. Roger Bumgarner Highly reproducible technical replicates

Fractional Fold Change 1. 1.5x dow n 67, 15, 2ng Total RNA Counts 1. 1. 1. 1. 1.5x up 2x up y = 1.965x -.467 R² =.998 y = 1.5x - 1.21 R 2 =.999 y =.676x -.551 R 2 =.998.1 1 1 1 1 1 1ng Total RNA Counts

Fractional Fold Change 2 1,8 1,6 1,4 1,2 1,8,6,4,2 * * * * * * * * * 1fM 1fM 1fM * p<.5 vs. 1. 1 1.2 1.4 1.6

Correlation with qpcr Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 7 16 18 1 1 16 25 12 45 45 6 5 4 3 2 1 EGG 9.3h 18h 24h 33h 48h 7h 14 12 1 8 6 4 2 16 14 12 1 8 6 4 2 EGG 9.3h 18h 24h 33h 48h 7h 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 EGG 9.3h 18h 24h 33h 48h 7h 14 12 1 8 6 4 2 2 15 1 5 EGG 9.3h 18h 24h 33h 48h 7h 1 8 6 4 2 4 35 3 25 2 15 1 5 EGG 9.3h 18h 24h 33h 48h 7h 4 35 3 25 2 15 1 5 Gene 6 Gene 7 Gene 8 Gene 9 Gene 1 6 2 3 7 25 45 1 2 4 35 5 4 3 2 1 18 16 14 12 1 8 6 4 2 25 2 15 1 5 6 5 4 3 2 1 2 15 1 5 4 35 3 25 2 15 1 5 9 8 7 6 5 4 3 2 1 18 16 14 12 1 8 6 4 2 35 3 25 2 15 1 5 3 25 2 15 1 5 EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h Gene 11 Gene 12 Gene 13 Gene 14 Gene 15 12 8 3 25 1 4 45 12 16 9 1 8 6 4 2 7 6 5 4 3 2 1 25 2 15 1 5 2 15 1 5 9 8 7 6 5 4 3 2 1 35 3 25 2 15 1 5 4 35 3 25 2 15 1 5 1 8 6 4 2 14 12 1 8 6 4 2 8 7 6 5 4 3 2 1 EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h Gene 16 Gene 17 Gene 18 Gene 19 Gene 2 4 18 1 18 3 9 25 45 14 3 35 3 25 2 15 1 5 EGG 9.3h 18h 24h 33h 48h 7h 16 14 12 1 8 6 4 2 9 8 7 6 5 4 3 2 1 EGG 9.3h 18h 24h 33h 48h 7h 16 14 12 1 8 6 4 2 25 2 15 1 5 EGG 9.3h 18h 24h 33h 48h 7h 8 7 6 5 4 3 2 1 2 15 1 5 EGG 9.3h 18h 24h 33h 48h 7h 4 35 3 25 2 15 1 5 12 1 8 6 4 2 EGG 9.3h 18h 24h 33h 48h 7h 25 2 15 1 5 Data Courtesy of Dr. Eric Davidson 2 genes across 7 time points in quadruplicates: 2 x 7 x 4 = 56 qpcr reactions 7 x 4 = 28 NanoString reactions NanoString QPCR

Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

Flexibility in Sample Input Total RNA Amplified RNA from Small Amount of Sample Whole Cell Lysates PaxGene Lysed Whole Blood Total RNA Extracted from FFPE Samples

Amplified vs. Unamplified Total RNA Log2 Fold Change 5ng BR/HR Amp plified Total RNA R² =,949 8, 6, 4, 2,, -8, -6, -4, -2,, 2, 4, 6, 8, -2, -4, -6, -8, Log2 Fold Change 1ng BR/HR Unamplified Total RNA Human Reference and Brain Reference RNA were used to generate Fold Change Data

Cell Lysate and Matched Total RNA Counts in Lysate Counts in Total RNA (1 ng) www.nanostring.com

Lysed vs Purified PaxGene Blood Data 16 14 12 Blood Lysate Prep 1 Blood Lysate Prep 2 Lysed Bl lood 1 8 6 4 y =,976x +,337 R² =,983 y =,985x +,451 R² =,987 2-2 2 4 6 8 1 12 14 16-2 Purified Total RNA No globin mitigation

FFPE & Frozen vs. Fresh RNA FFPE & Frozen Tissue Derived Total RNA 12 1 8 6 4 2-2 Heart total vs frozen Heart total vs FFPE y =,98x +,13 R² =,928 y =,883x -,48 R² =,925-2 2 4 6 8 1 12 Fresh Tissue Derived Total RNA

Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

Ewing s Sarcoma Fusion Transcripts Wild-type EWS Wild-type FLI EWS exon 7-FLI exon 5 fusion EWS exon 5-FLI exon 5 fusion EWS exon 7-FLI exon 6 fusion

Ewing s Sarcoma Fusion Transcripts B EWS exon 7 capture probe; FLI exon 5 reporter probe B B

Ewing s Sarcoma Fusion Transcripts B EWS exon 7 capture probe; FLI exon 6 reporter probe B B

Ewing s Sarcoma Fusion Transcripts B EWS exon 7 capture probe; FLI exon 6 reporter probe B B B

Ewing s Sarcoma Fusion Transcripts Experiment: Prepare RNA (or lysate) from tumor samples Hybridize to CodeSet designed against specific breakpoints Determine which of the breakpoint-specific probes predominates Prediction: since tumor is likely to be monoclonal, only one of the transcripts will be present at high concentrations

Ewing s Sarcoma Fusion Transcripts Transfected cells EWS Cell Line A EWS Cell Line B EWS Cell Line C EWS Cell Line D EWS Cell Line E EWS Cell Line F EWS Tumor A EWS Tumor B FFPE Tumor A FFPE Tumor B 1% Concordance with known breakpoints

Ewing s Sarcoma Fusion Transcripts Data courtesy of Marc Ladanyi, MD

ncounter Analysis System Very simple & automated assay Direct measurement of target No enzymes; No RT; No amplification Digital data 1 code count = 1 mrna Sensitive, linear, quantitative & reproducible Up to 576 genes measured in a single tube Over 4,32 data points can be generated in 24 hours (72 samples x 56 genes) No sample preparation

ncounter TM Analysis System Molecules That Count TM www.nanostring.com