Post on 31-Mar-2015
Multiplex digital nucleic acid quantitation using molecular barcodes
Paul Rasmussen
Sr. Manager of Emerging Markets and Consumable Programs
prasmussen@nanostring.com
NanoString Confidential.1
NanoString Confidential.2
Agenda
Platform Introduction
Chemistry Overview
Performance
Application extensions miRNA and CNVs
NanoString nCounter assay: Single reaction, up to 800 targets
QPCR
Microarrays/NGS
Sens
itivity
Multiplexing # of Transcripts
NanoString
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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 precisionAccess 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
2008 2009 2010 2011 20120
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projected by EOY
pubs per year
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
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nCounter Prep Station
nCounter Digital Analyzer
NanoString Confidential.6
Agenda
Platform Introduction
Chemistry Overview
Performance
Application extensions miRNA and CNVs
Each Barcode Attached to an Individual RNA
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
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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
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nCounter CodeSet
Pre-mixed sets of all probes and controls
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Capture Probes
Reporter Probes
System Controls
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Day 2AUTOMATED
The nCounter Assay: Three Simple Steps
5 minHANDS-ON
Day 2AUTOMATED
5 minHANDS-ON
Day 15 minHANDS-ON
Purify2
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nCounter Prep Station
nCounter Digital Analyzer
Hybridize1
Flexible sample requirements
Only 4 pipetting steps
No amplification
800 hybridizations in single tube
Count3
Sensitive
Precise
Quantitative
Simple
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
HybridizeCodeSet to RNA
Count codes
nCounter Assay
mRNA Capture & Reporter Probes
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Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
HybridizeCodeSet to RNA
Count codes
nCounter Assay
Hybridized mRNA Excess Reporters
13 NanoString Confidential.
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
HybridizeCodeSet to RNA
Count codes
nCounter Assay
Hybridized Probes Bind to Cartridge
Surface of cartridge is coated with streptavidin
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Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
HybridizeCodeSet to RNA
Count codes
nCounter Assay
Immobilize and align reporter for image collecting and barcode counting
15 NanoString Confidential.
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
HybridizeCodeSet to RNA
Count codes
nCounter Assay
Image Surface
One coded reporter = 1 mRNA
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Code Gene Countx 3
y 1
z 2
nCounter Assay
Codes are counted and tabulated
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
HybridizeCodeSet to RNA
Count codes
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Simple read out of counts
NanoString Confidential.19
Agenda
Platform Introduction
Chemistry Overview
Performance
Application extensions miRNA and CNVs
The nCounter Assay: Very Reproducible
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R2 = 0.9999
Repl
icate
1 C
ount
sReproducibility of NanoString Assay Technical Replicates
Replicate 2 Counts
Data Courtesy of Dr. Roger Bumgarner
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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 1 Site 2
Site 3
R2 Site1 v Site 2 = 0.97R2 Site1 v Site 3 = 0.98
NanoString® Technologies | Confidential23
Very good cross platform performance: qPCR
Khan et al., 2011
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
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
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
NanoString® Technologies | Confidential27
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
Formalin fixation inhibits qPCR much more than the nCounter platform
Heart Frozen/FFPE
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POLR2a LDHA GRPR C13orf23 NDUFV3
PCRnCounter
Fold
Dec
reas
e
NanoString Confidential.29
Unparalleled Performance on FFPE Samples
mRNA Transcript Quantification in Archival Samples Using Multiplexed, Color-coded ProbesReis, 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)
NanoString Confidential.30
Sample flexibility: Cell Lysate and Matched Total RNA
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f(x) = 0.298104770543451 x + 7.194209916585R² = 0.976173007157184
f(x) = 0.630093248384163 x + 12.3939054285541R² = 0.977808883097132
f(x) = 1.30173923776692 x + 22.2432014725715R² = 0.972857263216265
10,000 cells vs 100ngLinear (10,000 cells vs 100ng)5,000 cells vs 100ngLinear (5,000 cells vs 100ng)2,500 cells vs 100ngLinear (2,500 cells vs 100ng)
Counts in 100ng RNA
Coun
ts in
Lys
ate
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
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PaxGene Blood Lysates vs. Purified RNA
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f(x) = 0.985428679843974 x + 0.451779490446374R² = 0.987902256477999f(x) = 0.976041231786931 x + 0.337161048855161R² = 0.983418605725623
Blood Lysate Prep 1Linear (Blood Lysate Prep 1)
log2 counts from total RNA purified from blood
log2
cou
nts
from
blo
od ly
sate
Measurements with unpurified PAXgene blood lysates correlate extremely well with purified RNA
NanoString Confidential.33
Agenda
Platform Introduction
Chemistry Overview
Performance
Application extensions miRNA, and CNVs
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nCounter miRNA Assays
Human miRNA Panel
Detects:800 human miRNAs3 nonhuman miRNAs (possible spike in controls)
Mouse miRNA Panel
Detects:578 mature mouse miRNAs33 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 ng purified total RNA
Linear Dynamic Range: 2106 counts
Hands on Time: <2 hours
NanoString Confidential.35
Current challenges to miRNA detection
Short lengthoverall low Tmprohibits 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
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1.00%
2.00%
3.00%
4.00%
5.00%
6.00%
Human miRNA Tm Distribution
Tm
% H
uman
miR
NA
s
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miRNA Sample Preparation Basics
miRNAs
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miRNA Sample Preparation Basics
miRNAs
Hybridize bridge oligoto each miRNA target
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miRNA Sample Preparation Basics
miRNAs
Bridge oligo specifically annealsto each miRNA target
Unique miRtag for each miRNA species
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miRNA Sample Preparation Basics
miRNA is covalently linked to miRtag via ligationmiRNAs
Bridge oligo specifically annealsto each miRNA target
Unique miRtag for each miRNA species
miRNA Sample Preparation Basics
Excess bridges and tags are removed
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Probe Architecture
Target Specific Capture & Reporter Probes bind to the chimaeric miRNA:miRtag molecule
Target Specific Capture Probe
Target Specific Reporter Probe
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Biotin
NanoString® Technologies | Confidential42
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 AnalysisUnambiguous Discrimination of miRNAs with
Single Nucleotide Differences
Dynamic range of the miRNA assay
Dynamic range of the miRNA assay
hsa-miR-1 expression in different tissues
New miRNA assays for the nCoutner assay
A la carte miRNAsSelect from 20-50 miRNAs from our panels for focused profilingWorkflow identical to standard miRNA assayUsers specify housekeepers (stable miRNAs for normalization)
miRGE assayMixed mRNA and miRNA codesets10-30 miRNAs from our panelsUp to 200 mRNAsWorkflow similar to our standard miRNA assay
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R² = 0.988600727780784
GEx vs miRGE assay format probe comparison
Counts for GEx format probes
Cou
nts
for m
iRG
E fo
rmat
pro
bes
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nCounter Copy Number Assay – Sample Preparation
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Genomic DNA Fragment ds genomic DNA using 4 base cutter (Alu 1)
Average~ 500 bases
Perform nCounter
Hybridization
Denature DNA @ 95C
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
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Calls agree with HapMap
One copy No copies
No copies
HapMap CNV nCounter CNV
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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
Very good correlation with HapMap
NanoString Confidential.50
The importance of Karyotyping….
Normal Female
HeLa
Molecules That CountTM
Paul Rasmussenprasmussen@nanostring.com
Thank You!