Multiplex digital nucleic acid quantitation using molecular barcodes
Download
1 / 50

Multiplex digital nucleic acid quantitation using molecular barcodes - PowerPoint PPT Presentation


  • 126 Views
  • Uploaded on

Multiplex digital nucleic acid quantitation using molecular barcodes. Paul Rasmussen Sr. Manager of Emerging Markets and Consumable Programs prasmussen@nanostring.com. Agenda. Platform Introduction Chemistry Overview Performance Application extensions miRNA and CNVs.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Multiplex digital nucleic acid quantitation using molecular barcodes' - efrat


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

prasmussen@nanostring.com

NanoString Confidential.


Agenda
Agenda barcodes

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNA and CNVs

NanoString Confidential.


Nanostring ncounter assay single reaction up to 800 targets
NanoString barcodesnCounter 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 barcodesnCounter 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 barcodesnCounter 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 barcodes

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNA and CNVs

NanoString Confidential.



Two probe assay
Two Probe Assay barcodes

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 barcodes

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 barcodes

Pre-mixed sets of all probes and controls

Capture Probes

System Controls

Reporter Probes

NanoString Confidential.


The ncounter assay three simple steps
The barcodesnCounter 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 barcodes

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 barcodes

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 barcodes

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 barcodes

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 barcodes

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 barcodes

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 barcodes

NanoString Confidential.


Agenda2
Agenda barcodes

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNAand CNVs

NanoString Confidential.


The ncounter a ssay very r eproducible
The barcodesnCounterAssay: 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 barcodes

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 barcodes: qPCR

Khan et al., 2011


Multiplex digital nucleic acid quantitation using molecular barcodes

Very good barcodescross 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 barcodesAffymetrix 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 barcodescross 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 barcodes

  • 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 barcodesqPCR much more than the nCounter platform

Fold Decrease


Multiplex digital nucleic acid quantitation using molecular barcodes

Unparalleled Performance on FFPE Samples barcodes

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 barcodes

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 barcodes

Khan et al., 2011

NanoString Confidential.


Multiplex digital nucleic acid quantitation using molecular barcodes

PaxGene Blood Lysates vs. Purified RNA barcodes

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 barcodes

  • Platform Introduction

  • Chemistry Overview

  • Performance

  • Application extensions miRNA, and CNVs

NanoString Confidential.


Ncounter mirna assays
nCounter barcodesmiRNA 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 barcodes

  • 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 barcodes Sample Preparation Basics

miRNAs


Mirna sample preparation basics1
miRNA barcodes Sample Preparation Basics

Hybridize bridge oligo

to each miRNA target

miRNAs


Mirna sample preparation basics2
miRNA barcodes Sample Preparation Basics

Bridge oligo specifically anneals

to each miRNA target

Unique miRtag for each miRNA species

miRNAs


Mirna sample preparation basics3
miRNA barcodes 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 barcodes Sample Preparation Basics

Excess bridges and tags are removed


Probe architecture
Probe Architecture barcodes

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

Biotin

Target Specific Capture Probe

Target Specific Reporter Probe


Ncounter mirna analysis
nCounter barcodesmiRNA 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 barcodesmiRNA assay


Dynamic range of the mirna assay1
Dynamic range of the barcodesmiRNA assay

hsa-miR-1 expression in different tissues


New mirna assays for the ncoutner assay
New barcodesmiRNA 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 barcodes 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 barcodes

  • 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 barcodesHapMap

One copy

No copies

No copies

nCounter CNV

HapMap CNV

NanoString Confidential.


Multiplex digital nucleic acid quantitation using molecular barcodes

Very good correlation with barcodesHapMap

  • 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…. barcodes

Normal Female

HeLa

NanoString Confidential.


Thank you
Thank You! barcodes

Molecules That CountTM

Paul Rasmussen

prasmussen@nanostring.com