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Multiplex digital nucleic acid quantitation using molecular barcodes Paul Rasmussen Sr. Manager of Emerging Markets and Consumable Programs

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Presentation on theme: "Multiplex digital nucleic acid quantitation using molecular barcodes Paul Rasmussen Sr. Manager of Emerging Markets and Consumable Programs"— Presentation transcript:

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

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

3 NanoString nCounter assay: Single reaction, up to 800 targets QPCR Microarrays/ NGS Sensitivity Multiplexing # of Transcripts NanoString 3NanoString Confidential ,000

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 4

5 The nCounter Analysis System: Two fully automated instruments Fully Automated 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 5 nCounter Prep Station nCounter Digital Analyzer

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

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 8NanoString 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 9

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

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

12 nCounter Assay mRNACapture & Reporter Probes 12NanoString Confidential.

13 nCounter Assay Hybridized mRNAExcess Reporters 13NanoString Confidential.

14 nCounter Assay Hybridized Probes Bind to Cartridge Surface of cartridge is coated with streptavidin 14NanoString Confidential.

15 nCounter Assay Immobilize and align reporter for image collecting and barcode counting 15NanoString Confidential.

16 nCounter Assay Image Surface One coded reporter = 1 mRNA 16NanoString Confidential.

17 CodeGeneCount x3 y1 z2 nCounter Assay Codes are counted and tabulated 17NanoString Confidential.

18 18 Simple read out of counts

19 Agenda Platform Introduction Chemistry Overview Performance Application extensions miRNA and CNVs NanoString Confidential.19

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

21 Superior Precision in Site-to-Site Reproducibility Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastoma samples Northcott P.E. et al., Acta Neuropathologica; November 16, 2011 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 1Site 2 Site 3 R 2 Site1 v Site 2 = 0.97 R 2 Site1 v Site 3 = 0.98

22 Very good cross platform performance: TaqMan qPCR 6 NanoString reactions, 372 PCR reactions PCR data from: Canales et al. Evaluation of DNA microarray results with quantitative gene expression, Nature Biotechnology 24, (2006) Log2 Fold Change 100ng BR/HR Total RNA nCounter Log2 Fold Change BR/HR TaqMan 22NanoString Confidential.

23 NanoString ® Technologies | Confidential 23 Very good cross platform performance: qPCR Khan et al., 2011

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

25 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

26 Very good cross platform performance: RNA-Seq NanoString Confidential.26 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

27 NanoString ® Technologies | Confidential 27 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 PaxGene Lysed Whole Blood Total RNA and DNA Extracted from FFPE Samples Crude Extracts from FFPE samples Plasma, Serum and other Biofluids

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

29 NanoString Confidential.29 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 … 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. nCounter ® (r = 0.90)qPCR (r = 0.50)

30 NanoString Confidential.30 Sample flexibility: Cell Lysate and Matched Total RNA Measurements with crude whole cell lysates correlate extremely well with purified RNA

31 NanoString Confidential.31 Flexibility of sample input Total RNA vs Lysate Khan et al., 2011

32 NanoString Confidential.32 PaxGene Blood Lysates vs. Purified RNA log2 counts from total RNA purified from blood log2 counts from blood lysate Measurements with unpurified PAXgene blood lysates correlate extremely well with purified RNA

33 Agenda Platform Introduction Chemistry Overview Performance Application extensions miRNA, and CNVs NanoString Confidential.33

34 nCounter miRNA 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 34 All miRNA Panels Sample Types Supported:RNA from Fresh/frozen tissue, FFPE, Blood, Cells Sample Input Recommendation:100 ng purified total RNA Linear Dynamic Range: counts Hands on Time:<2 hours

35 NanoString Confidential.35 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

36 miRNA Sample Preparation Basics 36 miRNAs

37 miRNA Sample Preparation Basics 37 miRNAs Hybridize bridge oligo to each miRNA target

38 miRNA Sample Preparation Basics 38 miRNAs Bridge oligo specifically anneals to each miRNA target Unique miRtag for each miRNA species

39 miRNA Sample Preparation Basics 39 miRNA is covalently linked to miRtag via ligation miRNAs Bridge oligo specifically anneals to each miRNA target Unique miRtag for each miRNA species

40 miRNA Sample Preparation Basics Excess bridges and tags are removed 40

41 Probe Architecture Target Specific Capture & Reporter Probes bind to the chimaeric miRNA:miRtag molecule Target Specific Capture Probe Target Specific Reporter Probe 41 Biotin

42 NanoString ® Technologies | Confidential 42 nCounter miRNA Analysis Multiplexed target profiling of miRNA transcriptomes in a single reaction Available for human, mouse, rat and drosophila High level of sensitivity, specificity, precision, and linearity nCounter ® miRNA Analysis Unambiguous Discrimination of miRNAs with Single Nucleotide Differences

43 Dynamic range of the miRNA assay

44 hsa-miR-1 expression in different tissues

45 New miRNA assays for the nCoutner assay A la carte miRNAs Select from 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 miRNA codesets miRNAs from our panels Up to 200 mRNAs Workflow similar to our standard miRNA assay NanoString Confidential.45

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

47 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 47NanoString Confidential.

48 Calls agree with HapMap NanoString Confidential.48 One copy No copies HapMap CNV nCounter CNV

49 Experiment: 100 HapMap samples + reference assayed (600ng digest/300ng hyb) 20 CNV regions with 3 probes analyzed. NanoString Confidential.49 Metric% of totalData points Call rate94.8%1902/2006 Accuracy (concordance)94.2%1791/1902 Very good correlation with HapMap

50 The importance of Karyotyping…. NanoString Confidential.50 Normal Female HeLa

51 Molecules That Count TM Paul Rasmussen Thank You!


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