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

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

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

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
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 5

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

7. Each Barcode Attached to an Individual RNA7

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. 8

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
System Controls
Target specific Capture probes and reporter probes are combined to create your custom CodeSet. To reiterate, NanoString builds the barcodes and assigns each gene sequence a unique barcode. Your CodeSet can contain target specific capture and reporter combinations sufficient for genes of your choice in a single tube. Both capture probes and reporter probes are in excess to drive the hybridization reaction to completion.
Each CodeSet is synthesized with dozens of internal controls included that allow for assessing the performance of each step in the process and for quantification of your endogenous transcripts. There is no need to waste extra reagents or sample material running extra controls like you would have to using qPCR, they are already included in each codeset.
Customers may opt to have additional endogenous genes included for normalization.
Sales Reps - Discuss the controls in depth later in the presentation during data.
Reporter Probes
NanoString Confidential. 10

11. The nCounter Assay: Three Simple Steps
5 min HANDS-ON
5 min HANDS-ON
5 min HANDS-ON
Day 1
Day 2
AUTOMATED
Day 2
AUTOMATED
nCounter Prep Station
nCounter Digital Analyzer
Hybridize 1
Flexible sample requirements
Only 4 pipetting steps
No amplification
800 hybridizations in single tube
Purify 2
Count 3
Sensitive
Precise
Quantitative
Simple
The protocol could not be more simple.
You add buffer, your CodeSet and sample into a strip tube and hybridize overnight.
Once hybridized, you simply load your hybridized samples, the nCounter Prep Plates and the nCounter Sample Cartridge on to the nCounter Prep Station.
Each run processes a single sample cartridge, or 12 samples. This takes about 2.5 hours.
After processing, you transfer your sample cartridge to the nCounter Digital Analyzer for imaging and data collection. The image processing and data collection take about 20 minutes per sample. You can load six cartridges on the analyzer and let it run overnight to process up to 72 samples unattended.
Both the nCounter Prep Station and the nCounter Digital Analyzer have easy-to-use touch screens that walk you through each step of the process. Everything you need to process the nCounter gene expression assay is available in a master kit. This includes all reagents and consumable plasticware (comprised of 3 sub-kits that have different storage requirements.)
NanoString Confidential. 11

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

13. nCounter Assay Hybridized mRNA Excess Reporters Hybridize
CodeSet to RNA
Remove
excess
reporters
Bind
reporter
to surface
Immobilize and align reporter
Image surface
Count codes
After the samples are hybridized, all of the sample processing steps are automated on the nCounter Prep Station.
The Prep Station uses a magnetic bead purification to isolate only Reporter Probes that have found a target. The excess CodeSet is washed away.
Hybridized mRNA
Excess Reporters
NanoString Confidential. 13

14. Hybridized Probes Bind to Cartridge
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. 14

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. 15

16. One coded reporter = 1 mRNA
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. 16

17. Codes are counted and tabulated
nCounter Assay
Hybridize
CodeSet 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
NanoString Confidential. 17

18. Simple read out of counts
NanoString Confidential.

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

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

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
Site 1
Site 2
Site 3
“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.”
The other major advantage I mentioned earlier is nCounter’s reproducibility.
These data are an excellent demonstration of this and were published last November by another group out of Toronto (at the Hospital for Sick Kids)
The authors used nCounter to validate a gene expression signature for medulloblastoma, but then they took there analysis one step further.
They shipped 48 RNA samples to 2 additional sites and performed an analysis of site-to-site reproducibility.
The heat maps show expression levels and patient categorization from each site
The scatterplot shows excellent correlation between the sites with R2 or .97 and .98 respectively when comparing Toronto to either of the 2 other sites
R2 Site1 v Site 2 = 0.97
R2 Site1 v Site 3 = 0.98

22. Very good cross platform performance: TaqMan qPCR
Log2 Fold Change 100ng BR/HR Total RNA nCounter
Log2 Fold Change BR/HR TaqMan
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)
NanoString Confidential. 22 22

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

24. Very good cross platform performance: Affymetrix
6 calibration genes were selected based on their known expression profiles in each myeloid cell fraction (CD34+ (2), promyelocyte (5), neutrophils(2)), M3 AML subtypes (11) and other AML subtypes (17). Both NanoString and Affymetrix data are shown. 28 samples were run in total (in parenthesis above). Data normalized to the index group (shown by asterisk).
Early Myeloid-specific heatopietic genes
Promyelocyte-specific genes
Late myeloid-specifici genes
Take hime message- On both a signature wide (33 genes) and individual gene basis, NanoString showed good correlation to the initial microarray data
Payton et al, High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples. J Clin Invest. June 2009

25. PCA of signature on Affymetrix and NanoString
Ultimately, the real litmus test is the signature’s ability to hold up in differentiating subtypes. The Principle component analysis above shows the original affy data (upper left), A subset of the original affy sample set on NanoString( upper right) and two validation cohorts on NanoString (bottom). Note that there is good separation of the M3 subtype (red) from M), M1, M2, and M4 subtypes (grey).
Also in the lower left, note the yellow samples. These were PML-RARA positive M3 samples initially missed by traditional cytogenetics. The green sample was morphologically called M2, but contains the PML-RARA translocation, and is classified as such in the sample set.
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
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.

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. 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.

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

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

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

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

34. nCounter miRNA Assays Human miRNA Panel Mouse miRNA Panel
Rat miRNA Panel
Detects:
800 human miRNAs
578 mature mouse miRNAs
423 mature rat miRNAs
3 nonhuman miRNAs (possible spike in controls)
33 mature murine-associated viral miRNAs
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: 2106 counts
Hands on Time: <2 hours

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
NanoString Confidential.

36. miRNA Sample Preparation Basics

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

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

39. 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

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

41. Probe Architecture
Target Specific Capture & Reporter Probes bind to the chimaeric miRNA:miRtag molecule
Biotin
The target sequence for the Capture Probe and the Reporter probe are designed to be adjacent to one another along a transcript.
Our probe design process picks probes with no gap or a one base pair gap in between the Capture Probe and Reporter Probe.
Probes are selected to span exon-exon boundries if possible.
Genomic DNA contamination has not been an issue in the nCounter system. The system never reaches denaturing conditions when used for RNA. Crossing exon boundaries also serves to eliminate the possibility of genomic contamination. Additionally the total RNA purification further reduces the potential. All things combined lead to no discernible genomic contamination in our hands. We have data further on in the presentation that shows empirical evidence of this (cell lysate slide).
With regard to gene families, We default in selection to capture the most transcripts possible. If there are several transcripts selected that are closely related in a gene family, a single probe will be selected to represent as many transcripts as possible in that family.
After the capture probe and reporter probe are bound to the mRNA, a stable tripartite structure is formed.
Target Specific Capture Probe
Target Specific Reporter Probe 41

42. nCounter miRNA Analysis
Unambiguous Discrimination of miRNAs with Single Nucleotide Differences
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 kits enable precise analysis of comprehensive collections of miRNAs from human, mouse and rat. To illustrate our ability to distinguish miRNAs that differ by a single nucleotide, we looked at the let7 family of highly related miRNAs. We synthesized probes against the 8 different let7 family members, and examined each for their reactivity with designated target, as well as cross-reactivity against other let7 familiy members.
Because we use a ligation-based method to extend the length of miRNAs, we can design the bridges and tags to exploit mismatches (they won’t ligate) and as a result there is little to no cross-reactivity,,

43. Dynamic range of the miRNA assay
100ng commercial purified RNA was analyzed with a single codeset detecting 676 miRNAs. Counts shown here are spike-normalized, singlet measurements. Counts were recovered over a dynamic range of 4-5 orders of magnitude. The two tissue types analyzed here are similar in origin (muscle-derived), so the high correlation (0.823) might reflect similar overall patterns of miRNA expression. Note howver that there are clearly differences in abundance of some miRNAs in the two muscle types.

44. Dynamic range of the miRNA assay
hsa-miR-1 expression in different tissues
Tissue-specific detection of a muscle-specific miRNA.

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
10-30 miRNAs from our panels
Up to 200 mRNAs
Workflow similar to our standard miRNA assay
NanoString Confidential.

46. nCounter Copy Number Assay – Sample Preparation
Perform nCounter Hybridization
Prior to hybridization, genomic DNA is fragmented and denatured. After that, the hybridization steps are the same as for other nCounter assays.
Genomic DNA
Fragment ds genomic DNA using 4 base cutter (Alu 1)
Average~ 500 bases
Denature 95C 46
NanoString Confidential.
NanoString Confidential.

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
NanoString Confidential.

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

49. Accuracy (concordance)
Very good correlation with HapMap
Experiment: 100 HapMap samples + reference assayed (600ng digest/300ng hyb)
20 CNV regions with 3 probes analyzed.
Metric
% of total
Data points
Call rate
94.8%
1902/2006
Accuracy (concordance)
94.2%
1791/1902
NanoString Confidential.

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

51. Molecules That CountTM
Thank You!
Molecules That CountTM
Paul Rasmussen 51