3 NanoString nCounter assay: Single reaction, up to 800 targets QPCRNanoStringSensitivityMicroarrays/NGS.,000Multiplexing # of TranscriptsNanoString 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 PNASPublications span most major disciplines in molecular biologyCancer, Immunology, Stem Cells, Systems Biology, AgricultureDriven by performance with FFPE and remarkable precisionAccess previously unusable samplesObserve biology not previously possiblePublication 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 StationnCounter Digital AnalyzerFully Automated sample processingUp to 800 genes per sample12 samples processed in one cartridgeUp to 4 cartridges per dayFully automated imaging and countingUp to 6 cartridges (72 samples) per day24 hour unattended processingSimple data output55
8 Two Probe AssayTarget Specific Capture & Reporter Probes are created to bind to the mRNA transcriptBiotinTarget Specific Capture ProbeTarget Specific Reporter ProbeNanoString Confidential.8
9 Both probes must hybridize Target Specific Capture & Reporter Probes are created to bind to the mRNA transcriptTarget Specific Capture ProbeTarget Specific Reporter Probe9
10 nCounter CodeSet Pre-mixed sets of all probes and controls Capture ProbesSystem ControlsTarget 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 ProbesNanoString Confidential.10
11 The nCounter Assay: Three Simple Steps 5 min HANDS-ON5 min HANDS-ON5 min HANDS-ONDay 1Day 2AUTOMATEDDay 2AUTOMATEDnCounter Prep StationnCounter Digital AnalyzerHybridize1Flexible sample requirementsOnly 4 pipetting stepsNo amplification800 hybridizations in single tubePurify2Count3SensitivePreciseQuantitativeSimpleThe 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 AssayHybridizeCodeSet to RNARemoveexcessreportersBindreporterto surfaceImmobilize and align reporterImage surfaceCount codesmRNACapture & Reporter ProbesNanoString Confidential.12
13 nCounter Assay Hybridized mRNA Excess Reporters Hybridize CodeSet to RNARemoveexcessreportersBindreporterto surfaceImmobilize and align reporterImage surfaceCount codesAfter 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 mRNAExcess ReportersNanoString Confidential.13
14 Hybridized Probes Bind to Cartridge nCounter AssayHybridizeCodeSet to RNARemoveexcessreportersBindreporterto surfaceImmobilize and align reporterImage surfaceCount codesSurface of cartridge is coated with streptavidinHybridized Probes Bind to CartridgeNanoString Confidential.14
15 nCounter AssayHybridizeCodeSet to RNARemoveexcessreportersBindreporterto surfaceImmobilize and align reporterImage surfaceCount codesImmobilize and align reporter for image collecting and barcode countingNanoString Confidential.15
16 One coded reporter = 1 mRNA nCounter AssayHybridizeCodeSet to RNARemoveexcessreportersBindreporterto surfaceImmobilize and align reporterImage surfaceCount codesImage SurfaceOne coded reporter = 1 mRNANanoString Confidential.16
17 Codes are counted and tabulated nCounter AssayHybridizeCodeSet to RNARemoveexcessreportersBindreporterto surfaceImmobilize and align reporterImage surfaceCount codesCodeGeneCountx3y1z2Codes are counted and tabulatedNanoString Confidential.17
18 Simple read out of counts NanoString Confidential.
20 The nCounter Assay: Very Reproducible Reproducibility of NanoString Assay Technical ReplicatesR2 =Replicate 1 CountsHReplicate 2 CountsData Courtesy of Dr. Roger BumgarnerNanoString Confidential.2020
21 Superior Precision in Site-to-Site Reproducibility Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastoma samplesNorthcott P.E. et al., Acta Neuropathologica; November 16, 2011Site 1Site 2Site 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 siteThe scatterplot shows excellent correlation between the sites with R2 or .97 and .98 respectively when comparing Toronto to either of the 2 other sitesR2 Site1 v Site 2 = 0.97R2 Site1 v Site 3 = 0.98
22 Very good cross platform performance: TaqMan qPCR Log2 Fold Change 100ng BR/HR Total RNA nCounterLog2 Fold Change BR/HR TaqMan6 NanoString reactions, 372 PCR reactionsPCR data from:Canales et al. Evaluation of DNA microarray results with quantitative gene expression,Nature Biotechnology 24, (2006)NanoString Confidential.2222
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 genesPromyelocyte-specific genesLate myeloid-specifici genesTake hime message- On both a signature wide (33 genes) and individual gene basis, NanoString showed good correlation to the initial microarray dataPayton 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, 2011NanoString Confidential.
27 What samples are you using: flexible options Total RNA and DNA (100ng/300ng/sample)Amplified RNA from Small Amount of SampleLCM and single cell (in progress)Whole Cell LysatesPaxGene Lysed Whole BloodTotal RNA and DNA Extracted from FFPE SamplesCrude Extracts from FFPE samplesPlasma, 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 ProbesReis, P.P. et al., BMC Biotechnology; May 9, 2011nCounter® (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 RNANanoString Confidential.
31 Flexibility of sample input Total RNA vs Lysate Khan et al., 2011NanoString Confidential.
32 PaxGene Blood Lysates vs. Purified RNA Measurements with unpurified PAXgene blood lysates correlate extremely well with purified RNAlog2 counts from total RNA purified from bloodlog2 counts from blood lysateNanoString Confidential.
34 nCounter miRNA Assays Human miRNA Panel Mouse miRNA Panel Rat miRNA PanelDetects:800 human miRNAs578 mature mouse miRNAs423 mature rat miRNAs3 nonhuman miRNAs (possible spike in controls)33 mature murine-associated viral miRNAsAll miRNA PanelsSample Types Supported: RNA from Fresh/frozen tissue, FFPE, Blood, CellsSample Input Recommendation: 100 ng purified total RNALinear Dynamic Range: 2106 countsHands on Time: <2 hours
35 Current challenges to miRNA detection Short lengthoverall low Tmprohibits concurrent binding, e.g. nCounter dual probeHighly Related SequencesLarge sequence diversityleads to large Tm spread even though length distribution is fairly smallNanoString Confidential.
38 miRNA Sample Preparation Basics Bridge oligo specifically annealsto each miRNA targetUnique miRtag for each miRNA speciesmiRNAs
39 miRNA Sample Preparation Basics Bridge oligo specifically annealsto each miRNA targetUnique miRtag for each miRNA speciesmiRNA is covalently linked to miRtag via ligationmiRNAs
40 miRNA Sample Preparation Basics Excess bridges and tags are removed
41 Probe ArchitectureTarget Specific Capture & Reporter Probes bind to the chimaeric miRNA:miRtag moleculeBiotinThe 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 ProbeTarget Specific Reporter Probe41
42 nCounter miRNA Analysis Unambiguous Discrimination of miRNAs with Single Nucleotide DifferencesMultiplexed target profiling of miRNA transcriptomes in a single reactionAvailable for human, mouse, rat and drosophilaHigh level of sensitivity, specificity, precision, and linearitynCounter 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 tissuesTissue-specific detection of a muscle-specific miRNA.
45 New miRNA assays for the nCoutner assay A la carte miRNAsSelect from 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 assayNanoString Confidential.
46 nCounter Copy Number Assay – Sample Preparation Perform nCounter HybridizationPrior to hybridization, genomic DNA is fragmented and denatured. After that, the hybridization steps are the same as for other nCounter assays.Genomic DNAFragment ds genomic DNA using 4 base cutter (Alu 1)Average~ 500 basesDenature 95C46NanoString Confidential.NanoString Confidential.
47 Variable copies of X chromosome Feasibility initially demonstrated with cell lines containing 1, 2, 3, 4 and 5 X chromosomesRequires fragmentation and denaturation of genomic DNAThe accuracy (measured vs. expected values) obtained with the nCounter system is extremely highNanoString Confidential.
48 Calls agree with HapMap One copyNo copiesNo copiesHapMap CNVnCounter CNVNanoString Confidential.
49 Accuracy (concordance) Very good correlation with HapMapExperiment: 100 HapMap samples + reference assayed (600ng digest/300ng hyb)20 CNV regions with 3 probes analyzed.Metric% of totalData pointsCall rate94.8%1902/2006Accuracy (concordance)94.2%1791/1902NanoString Confidential.
50 The importance of Karyotyping…. Normal FemaleHeLaNanoString Confidential.
51 Molecules That CountTM Thank You!Molecules That CountTMPaul Rasmussen51