Multiplex digital nucleic acid quantitation using molecular barcodes

1. Multiplex digital nucleic acid quantitation using molecular barcodes Paul Rasmussen Sr. Manager of Emerging Markets and Consumable Programs prasmussen@nanostring.com NanoString Confidential.

2. Agenda • Platform Introduction • Chemistry Overview • Performance • Application extensions miRNA and CNVs NanoString Confidential.

3. NanoStringnCounter assay: Single reaction, up to 800 targets NanoString QPCR Sensitivity Microarrays/NGS 1 10 100 1000 10,000 Multiplexing # of Transcripts NanoString Confidential.

4. The nCounter platform facilitates powerful research • Over 100 papers have been published using the nCounter platform as of June-2012, at a rate more than doubling yearly • >20% are published in Science, Nature, Cell, or PNAS • Publications span most major disciplines in molecular biology • Cancer, Immunology, Stem Cells, Systems Biology, Agriculture • Driven by performance with FFPE and remarkable precision • Access previously unusable samples • Observe biology not previously possible • Publication rate underestimates utilization of the platform by large pharma and industry. • Often less motivated to publish

5. The nCounter Analysis System: Two fully automated instruments nCounter Prep Station nCounter Digital Analyzer • FullyAutomated sample processing • Up to 800 genes per sample • 12 samples processed in one cartridge • Up to 4 cartridges per day • Fully automated imaging and counting • Up to 6 cartridges (72 samples) per day • 24 hour unattended processing • Simple data output

6. Agenda • Platform Introduction • Chemistry Overview • Performance • Application extensions miRNA and CNVs NanoString Confidential.

7. Each Barcode Attached to an Individual RNA

8. Two Probe Assay Target Specific Capture & Reporter Probes are created to bind to the mRNA transcript Biotin Target Specific Capture Probe Target Specific Reporter Probe NanoString Confidential.

9. Both probes must hybridize Target Specific Capture & Reporter Probes are created to bind to the mRNA transcript Target Specific Capture Probe Target Specific Reporter Probe

10. nCounter CodeSet Pre-mixed sets of all probes and controls Capture Probes System Controls Reporter Probes NanoString Confidential.

11. The nCounter Assay: Three Simple Steps 5minHANDS-ON 5minHANDS-ON 5minHANDS-ON Day1 Day2 AUTOMATED Day2 AUTOMATED nCounter Prep Station nCounter Digital Analyzer Hybridize Count Purify 3 1 2 • Flexible sample requirements • Only 4 pipetting steps • No amplification • 800 hybridizations in single tube • Sensitive • Precise • Quantitative • Simple NanoString Confidential.

12. nCounter Assay Hybridize CodeSet to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Capture & Reporter Probes mRNA NanoString Confidential.

13. nCounter Assay Hybridize CodeSet to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Hybridized mRNA Excess Reporters NanoString Confidential.

14. nCounter Assay Hybridize CodeSet 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 NanoString Confidential.

15. nCounter Assay Hybridize CodeSet to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Immobilize and align reporter for image collecting and barcode counting NanoString Confidential.

16. nCounter Assay Hybridize CodeSet to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Image Surface One coded reporter = 1 mRNA NanoString Confidential.

17. nCounter Assay Hybridize CodeSet to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Codes are counted and tabulated NanoString Confidential.

18. Simple read out of counts NanoString Confidential.

19. Agenda • Platform Introduction • Chemistry Overview • Performance • Application extensions miRNAand CNVs NanoString Confidential.

20. The nCounterAssay: Very Reproducible Reproducibility of NanoString Assay Technical Replicates R2 = 0.9999 Replicate 1 Counts Replicate 2 Counts Data Courtesy of Dr. Roger Bumgarner NanoString Confidential.

21. Superior Precision in Site-to-Site Reproducibility Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastomasamples Northcott P.E. et al., Acta Neuropathologica; November 16, 2011 Site 1 Site 2 “We present an assay based on NanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials.” Site 3 R2Site1 v Site 2 = 0.97 R2Site1 v Site 3 = 0.98

22. Very good cross platform performance: qPCR Khan et al., 2011

23. Very good cross platform performance: Affymetrix Payton et al, High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples. J Clin Invest. June 2009

24. PCA of signature on Affymetrix and NanoString Payton et al, High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples. J Clin Invest. June 2009

25. Very good cross platform performance: RNA-Seq Sun et al. Integrated analysis of Gene Expression, CpG Island Methylation, and Gene Copy Number in Breast Cancer Cells by Deep Sequencing. PLoSone, Feb, 2011 NanoString Confidential.

26. What samples are you using: flexible options • Total RNA and DNA (100ng/300ng/sample) • Amplified RNA from Small Amount of Sample • LCM and single cell (in progress) • Whole Cell Lysates • PaxGeneLysed Whole Blood • Total RNA and DNA Extracted from FFPE Samples • Crude Extracts from FFPE samples • Plasma, Serum and other Biofluids

27. Formalin fixation inhibits qPCR much more than the nCounter platform Fold Decrease

28. Unparalleled Performance on FFPE Samples mRNA Transcript Quantification in Archival Samples Using Multiplexed, Color-coded Probes Reis, P.P. et al., BMC Biotechnology; May 9, 2011 nCounter®(r = 0.90) qPCR(r = 0.50) “… the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples.We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.” NanoString Confidential.

29. Sample flexibility: Cell Lysate and Matched Total RNA Measurements with crude whole cell lysates correlate extremely well with purified RNA NanoString Confidential.

30. Flexibility of sample input Total RNA vs Lysate Khan et al., 2011 NanoString Confidential.

31. PaxGene Blood Lysates vs. Purified RNA Measurements with unpurified PAXgene blood lysates correlate extremely well with purified RNA log2 counts from blood lysate log2 counts from total RNA purified from blood NanoString Confidential.

32. Agenda • Platform Introduction • Chemistry Overview • Performance • Application extensions miRNA, and CNVs NanoString Confidential.

33. nCountermiRNA Assays Human miRNA Panel • Detects: • 800 human miRNAs • 3 nonhuman miRNAs (possible spike in controls) Mouse miRNA Panel • Detects: • 578 mature mouse miRNAs • 33 mature murine-associated viral miRNAs Rat miRNA Panel • Detects: • 423 mature rat miRNAs All miRNA Panels Sample Types Supported: RNA from Fresh/frozen tissue, FFPE, Blood, Cells Sample Input Recommendation: 100 ngpurified total RNA Linear Dynamic Range: 2106 counts Hands on Time: <2 hours

34. Current challenges to miRNA detection • Short length • overall low Tm • prohibits concurrent binding, e.g. nCounter dual probe • Highly Related Sequences • Large sequence diversity • leads to large Tm spread even though length distribution is fairly small NanoString Confidential.

35. miRNA Sample Preparation Basics miRNAs

36. miRNA Sample Preparation Basics Hybridize bridge oligo to each miRNA target miRNAs

37. miRNA Sample Preparation Basics Bridge oligo specifically anneals to each miRNA target Unique miRtag for each miRNA species miRNAs

38. miRNA Sample Preparation Basics Bridge oligo specifically anneals to each miRNA target Unique miRtag for each miRNA species miRNA is covalently linked to miRtag via ligation miRNAs

39. miRNA Sample Preparation Basics Excess bridges and tags are removed

40. Probe Architecture Target Specific Capture & Reporter Probes bind to the chimaericmiRNA:miRtag molecule Biotin Target Specific Capture Probe Target Specific Reporter Probe

41. nCountermiRNA Analysis • Multiplexed target profiling of miRNAtranscriptomes in a single reaction • Available for human, mouse, rat and drosophila • High level of sensitivity, specificity, precision, and linearity Unambiguous Discrimination of miRNAs with Single Nucleotide Differences nCounter® miRNA Analysis

42. Dynamic range of the miRNA assay

43. Dynamic range of the miRNA assay hsa-miR-1 expression in different tissues

44. New miRNA assays for the nCoutner assay • A la carte miRNAs • Select from 20-50 miRNAs from our panels for focused profiling • Workflow identical to standard miRNA assay • Users specify housekeepers (stable miRNAs for normalization) • miRGE assay • Mixed mRNA and miRNAcodesets • 10-30 miRNAs from our panels • Up to 200 mRNAs • Workflow similar to our standard miRNA assay NanoString Confidential.

45. nCounter Copy Number Assay – Sample Preparation Perform nCounter Hybridization Genomic DNA Fragment ds genomic DNA using 4 base cutter (Alu 1) Average~ 500 bases Denature DNA @ 95C 46 NanoString Confidential. NanoString Confidential.

46. Variable copies of X chromosome • Feasibility initially demonstrated with cell lines containing 1, 2, 3, 4 and 5 X chromosomes • Requires fragmentation and denaturation of genomic DNA • The accuracy (measured vs. expected values) obtained with the nCounter system is extremely high NanoString Confidential.

47. Calls agree with HapMap One copy No copies No copies nCounter CNV HapMap CNV NanoString Confidential.

48. Very good correlation with HapMap • Experiment: 100 HapMap samples + reference assayed (600ng digest/300ng hyb) • 20 CNV regions with 3 probes analyzed. NanoString Confidential.

49. The importance of Karyotyping…. Normal Female HeLa NanoString Confidential.

50. Thank You! Molecules That CountTM Paul Rasmussen prasmussen@nanostring.com