Multiplex Assay Design

Geeta Bhat, Luminex Molecular Diagnostics; Toronto.

APHL/CDC Newborn Screening Molecular Workshop, CDC, Atlanta, GA June 28-30, 2011

Luminex

Multiplexed Solutions. For Life.

Luminex is the leader in microsphere-based multiplexing Luminex technology is used by researchers and physicians

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 ™ System Advanced 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

xMAP Technology: Data collection

Software sorts data by size scatter Events larger or smaller than microspheres are excluded Microsphere size is set by “gate” in each experiment

Larger

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

Designing a Universal Sequence Set

• • • 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

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

BUILD THE REST Universally-tagged, biotin-labelled representation of sample genomic DNA

B B Anti-Tag > Tag FOUNDATION IS BUILT

The xTAG

™

Genotyping Platform

5 Easy Steps

I. Multiplex PCR B B B II. Multiplex ASPE III.

Universal Array Sorting IV. xMAP ™ Detection B B PE B PE V. Data Analysis

Multiplex PCR

Ladder 500 bp 400 bp 300 bp 250 bp 200 bp 175 bp Sample: Neg 1 2 3 4 5

Single tube 16-plex PCR

Multiplex ASPE Genotyping

A llele S pecific P rimer E xtension

3’ 3’ Tag 1 3’ Tag 1 A T A T T 3’ PCR-amplified wt DNA 3’ Denature Anneal allele-specific tagged primers Tagged wt primer Tagged mut primer Tag 2 3’ 3’ A T A T Extend with DNA polymerase and biotin-dCTP B A T B Tag 2 3’ A No extension 3’ 3’

Universal Array Sorting

Tag/Anti-Tag 1 B SA B SA PE Tag/Anti-Tag 3 Tag/Anti-Tag 2 Universal array made up of n different bead populations

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 AR wt = 0.30-1.00

• • mut allele present homozygous wt AR mut AR wt = 0.30-1.00

= 0.85-1.00

Allelic Ratios (AR)

AR mut = Net MUT Signal Net MUT Signal + Net WT Signal

AR mut Values WT Heterozygous MUT 0 0.15 0.30 0.70 0.85 1.0

Assay Format Summary

5 Steps: I II III IV V Genomic DNA Whole blood Blood spots Mouthwash WGA Single tube Multiplex PCR Single tube Multiplex ASPE xTAG ™ Universal Array Sorting Data Acquisition on Luminex xMAP 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

Load & analyze data file(s) Open detailed views Print / Export analysis result Primary negative control Security Access Control Command Line Custom assay configurations (LDTC) 26

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 27

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) 28

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