Multiplex digital nucleic acid quantitation using molecular barcodes
This presentation is the property of its rightful owner.
Sponsored Links
1 / 50

Multiplex digital nucleic acid quantitation using molecular barcodes PowerPoint PPT Presentation


  • 88 Views
  • Uploaded on
  • Presentation posted in: General

Multiplex digital nucleic acid quantitation using molecular barcodes. Paul Rasmussen Sr. Manager of Emerging Markets and Consumable Programs [email protected] Agenda. Platform Introduction Chemistry Overview Performance Application extensions miRNA and CNVs.

Download Presentation

Multiplex digital nucleic acid quantitation using molecular barcodes

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Multiplex digital nucleic acid quantitation using molecular barcodes

Multiplex digital nucleic acid quantitation using molecular barcodes

Paul Rasmussen

Sr. Manager of Emerging Markets and Consumable Programs

[email protected]

NanoString Confidential.


Agenda

Agenda

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNA and CNVs

NanoString Confidential.


Nanostring ncounter assay single reaction up to 800 targets

NanoStringnCounter assay: Single reaction, up to 800 targets

NanoString

QPCR

Sensitivity

Microarrays/NGS

1 10 100 1000 10,000

Multiplexing # of Transcripts

NanoString Confidential.


The ncounter platform facilitates powerful research

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


The ncounter analysis system two fully automated instruments

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


Agenda1

Agenda

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNA and CNVs

NanoString Confidential.


Each barcode attached to an individual rna

Each Barcode Attached to an Individual RNA


Two probe assay

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.


Both probes must hybridize

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


Ncounter codeset

nCounter CodeSet

Pre-mixed sets of all probes and controls

Capture Probes

System Controls

Reporter Probes

NanoString Confidential.


The ncounter assay three simple steps

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.


Ncounter assay

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.


Ncounter assay1

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.


Ncounter assay2

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.


Ncounter assay3

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.


Ncounter assay4

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.


Ncounter assay5

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.


Multiplex digital nucleic acid quantitation using molecular barcodes

Simple read out of counts

NanoString Confidential.


Agenda2

Agenda

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNAand CNVs

NanoString Confidential.


The ncounter a ssay very r eproducible

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.


Superior precision in site to site reproducibility

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


Very good cross platform performance qpcr

Very good cross platform performance: qPCR

Khan et al., 2011


Multiplex digital nucleic acid quantitation using molecular barcodes

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


Pca of signature on affymetrix and nanostring

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

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.


What samples are you using flexible options

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


Formalin fixation inhibits qpcr much more than the ncounter platform

Formalin fixation inhibits qPCR much more than the nCounter platform

Fold Decrease


Multiplex digital nucleic acid quantitation using molecular barcodes

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.


Multiplex digital nucleic acid quantitation using molecular barcodes

Sample flexibility: Cell Lysate and Matched Total RNA

Measurements with crude whole cell lysates correlate extremely well with purified RNA

NanoString Confidential.


Multiplex digital nucleic acid quantitation using molecular barcodes

Flexibility of sample input Total RNA vs Lysate

Khan et al., 2011

NanoString Confidential.


Multiplex digital nucleic acid quantitation using molecular barcodes

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.


Agenda3

Agenda

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNA, and CNVs

NanoString Confidential.


Ncounter mirna assays

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


Multiplex digital nucleic acid quantitation using molecular barcodes

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.


Mirna sample preparation basics

miRNA Sample Preparation Basics

miRNAs


Mirna sample preparation basics1

miRNA Sample Preparation Basics

Hybridize bridge oligo

to each miRNA target

miRNAs


Mirna sample preparation basics2

miRNA Sample Preparation Basics

Bridge oligo specifically anneals

to each miRNA target

Unique miRtag for each miRNA species

miRNAs


Mirna sample preparation basics3

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


Mirna sample preparation basics4

miRNA Sample Preparation Basics

Excess bridges and tags are removed


Probe architecture

Probe Architecture

Target Specific Capture & Reporter Probes bind to the chimaericmiRNA:miRtag molecule

Biotin

Target Specific Capture Probe

Target Specific Reporter Probe


Ncounter mirna analysis

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


Dynamic range of the mirna assay

Dynamic range of the miRNA assay


Dynamic range of the mirna assay1

Dynamic range of the miRNA assay

hsa-miR-1 expression in different tissues


New mirna assays for the ncoutner assay

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.


Ncounter copy number assay sample preparation

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.


Variable copies of x chromosome

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.


Calls agree with hapmap

Calls agree with HapMap

One copy

No copies

No copies

nCounter CNV

HapMap CNV

NanoString Confidential.


Multiplex digital nucleic acid quantitation using molecular barcodes

Very good correlation with HapMap

  • Experiment: 100 HapMap samples + reference assayed (600ng digest/300ng hyb)

  • 20 CNV regions with 3 probes analyzed.

NanoString Confidential.


The importance of karyotyping

The importance of Karyotyping….

Normal Female

HeLa

NanoString Confidential.


Thank you

Thank You!

Molecules That CountTM

Paul Rasmussen

[email protected]


  • Login