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

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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!