multiplex assay design geeta bhat, luminex molecular diagnostics; toronto. aphl/cdc newborn...
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Multiplex Assay DesignGeeta Bhat, Luminex Molecular Diagnostics; Toronto.
APHL/CDC Newborn Screening Molecular Workshop, CDC, Atlanta, GA June 28-30, 2011
Luminex
Luminex is the leader in microsphere-based multiplexing
Luminex technology is used by researchers and physicians
Multiplexed Solutions. For Life.
Luminex – Multiplex Assay Design
Components of an multiplexed assay:
Platform
Technology
Data Analysis Software
What is xMAP® Technology?
Combination of multiple proven technologies
Color-coded 5.6 micron microspheres 100 different colors, proprietary dyeing process
Luminex® 100/200™ SystemAdvanced optics, lasers, fluidics, DSPs, software
The Luminex 100/200 is a class 1 (I) laser product.
The Luminex xMAP® Platform• Derived from classical flow cytometry• Faster hybridization kinetics due to quasi-solution phase reaction• Beads
• Polystyrene/Magnetic• 5.6/6.5 um diameter• uniform• dyed using a proprietary process
• Hardware is a simple benchtop flow analyzer• fluidics • optics• digital signal processor
• Multiplexible from 50 – 100 analytes• High throughput 96-well microplate format • Easily automatable• Flexible open platform (array size can vary)
How Does xMAP Technology Work?
• Microspheres are dyed to create 100 distinct colors
• Each microsphere has ‘spectral address’ based on red/infrared content
• Microspheres are suspendable• Microspheres are coated with capture
reagent (oligo or antibody)• Sample is added to microspheres• Analyte is captured to microspheres• Fluorescent reporter tag added
How Does xMAP Technology Work?
• Microspheres are dyed to create 100 distinct colors
• Each microsphere has ‘spectral address’ based on red/infrared content
• Microspheres are suspendable• Microspheres are coated with
capture reagent (oligo or antibody)• Sample is added to microspheres• Analyte is captured to
microspheres• Fluorescent reporter tag added
How Does xMAP Technology Work?
• Microspheres are dyed to create 100 distinct colors
• Each microsphere has ‘spectral address’ based on red/infrared content
• Microspheres are suspendable• Microspheres are coated with
capture reagent (oligo or antibody)• Sample is added to microspheres• Analyte is captured to
microspheres• Fluorescent reporter tag added
How Does xMAP Technology Work?
• Assays are read using a compact microsphere analyzer
• Analyzer samples well• Lasers excite fluorescent dyes- red
laser for bead classification and green for assay result
• Multiple readings for each microsphere set
• Software reports results in real-time• Up to 9600 results read in one hour
The Luminex 100/200 is a class 1 (I) laser product.
How Does xMAP Technology Work?
• Assays are read using a compact microsphere analyzer
• Analyzer samples well• Lasers excite fluorescent dyes- red
laser for bead classification and green for fluorescence detection
• Multiple readings for each microsphere set
• Software reports results in real-time• Up to 9600 results read in one hour
The Luminex 100/200 is a class 1 (I) laser product.
How Does xMAP Technology Work?
• Assays are read using a compact microsphere analyzer
• Analyzer samples well• Lasers excite fluorescent dyes- red
laser for bead classification and green for assay result
• Multiple readings for each microsphere set
• Software reports results in real-time• Up to 9600 results read in one hour
Larger
xMAP Technology: Data collection
Events larger or smaller than microspheres are excluded
Microsphere size is set by “gate” in each experiment
Software sorts data by size scatter
Designing a Universal Sequence Set
A combinatorial problem in that one must design N DNA tags such that:
• each tag hybridizes efficiently to its complementary anti-tag but not to any other of the N-1 anti-tags
• tags have similar melting temperature
• tags have similar lengths
• tags are not too similar to actual genes
The universal tags have to be different enough to be distinguishable from one another and the genome but similar enough to be able to control their behavior under a single set of conditions.
Tag = target; Anti-tag = probe
• All sequences will be 24mers • Pattern matching/similarity thresholds
• no common subsequence with melting temp. above some fixed threshold
• no alignment with score above some fixed threshold
• Result is sets of minimally cross-hybridizing isothermal sequences
Designing a Universal Sequence Set
Benefits of Universal Arrays
• Single array needs optimized only once for all applications• Compatible with multiple genotyping (front-end) chemistries• Simplified manufacturing, QC• Is a diagnostic industry standard
• Improved accuracy, S/N ratio
• Overcome back-end limits of multiplexing• Sequences have been DESIGNED to work together
• Dramatically decreases development time for new products (ie. Lower costs for assay development)
The xTAG Genotyping Platform
Anti-Tag Tag
>FOUNDATION IS BUILT
BUILD THE REST
Universally-tagged, biotin-labelled
representation of sample genomic DNA
B
B
I. Multiplex PCR
PEBPEB
BBB
B
II. Multiplex ASPE
III.
IV. xMAP™ Detection
Universal Array Sorting
The xTAG™ Genotyping Platform
V. Data Analysis
5 Easy Steps
Multiplex PCR
Sample: Neg 1 2 3 4 5
175 bp
200 bp
250 bp
300 bp
400 bp 500 bp
Ladder
Single tube 16-plex PCR
A3’
T 3’
Multiplex ASPE Genotyping
Denature Anneal allele-specific tagged primers
Extend with DNA polymerase and biotin-dCTP
TTag 1A3’
T 3’
TTag 1
CTag 2A3’
CTag 2A3’
PCR-amplified wt DNAA3’T 3’ T
A3’
3’
B
B
Allele Specific Primer Extension
Tagged wt primerTagged mut primer
No extension
Universal Array Sorting
Universal array made up of n different bead
populations
B SA PEPEB SA
Tag/Anti-Tag 3
Tag/Anti-Tag 1 ASPE rxn
Tag/Anti-Tag 2
PEPE
xTAG Genotype Calling & Allelic Ratios
• Genotypes based on the allelic ratio (AR) for mutation in question
• AR is calculated by expressing the net signal from a given allele as a fraction of the total signal (wt and mut) for each mutation analyzed
• Both wt and mut AR’s are calculated by TDAS to make calls
• AR ranges for genotype calling determined empirically for each mutation
• wt present ARwt = 0.30-1.00• mut allele present ARmut = 0.30-1.00• homozygous wt ARwt = 0.85-1.00
Allelic Ratios (AR)
Net MUT Signal
Net MUT Signal + Net WT SignalARmut =
WT Heterozygous MUT
0 0.15 0.30 0.70 0.85 1.0
ARmut Values
Assay Format Summary
Genomic DNA
Whole bloodBlood spotsMouthwash
WGA
Data
Acquisition
on Luminex
xMAP
Single
tube
Multiplex
PCR
Single
tube
Multiplex
ASPE
xTAG™
Universal
Array Sorting
5 Steps: I II III IV V
Data
Analysis
TDAS
Conclusions
The combination of a bead-based platform with the universal array principle provides several benefits:
• Less development time for new application• Higher accuracy (specificity in solution
phase)• Flexibility in array size• High throughput• Economical• Simplifies array manufacturing and QC• Open platform provides labs with an
opportunity to build their own assays
What is TDAS LSM?
Easy-to-use data analysis software for used with assays that are built on xTAG® technology
Provide qualitative calls for samples based on raw signals (MFI – median fluorescence intensity) acquired from the Luminex Analyzer.
User can customize the analysis for their assays:Organize analytes into targets and panels
Specify cut-offs for making calls
Define call dependency rules
25
TDAS LSM features
26
Load & analyze data file(s)
Open detailed views
Print / Export analysis result
Primary negative control
Security Access Control
Command Line
Custom assay configurations (LDTC)
27
TDAS LSM Analysis Module
Signal-Based Analysis (SBA) Module
Suitable for assays that have independent analytes for each target detection, e.g. infectious diseases, one analyte for each virus or bacteria
Analyze sample data based on the analyte (probe) signals for a target
Use MFI cutoffs to make calls
Support signal-to-noise ratio
Target can have one or multiple analytes
Calls are made on target only
28
TDAS LSM Analysis Module
Ratio-Based Analysis (RBA) Module
Suitable for human genetics assays that use multiple analytes to detect the presence or absence of the variants, e.g. wild-type and mutant allele
Analyze sample data based on the ratio of the analyte signals for a target
SAT – a Single Analyte Target can have either a wild-type analyte or a variant analyte
MAT – a Multiple Analyte Target could have
– at least one wild-type analyte and one variant analyte
– multiple analytes for variants only (MVT)
Thank You!
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