SNPlex™ Genotyping System:A New High Throughput Solution

What is the SNPlex™ System?

Assay based on multiplexed Oligonucleotide Ligation Assay (OLA) and PCR technology Highly specific High order multiplexing Robust

Utilizes universal assay reagents Ready to use components (includes buffers, enzymes and assay controls)-no

preparation required Streamline workflow Consistent, reproducible results

Compatible with common laboratory equipment Based upon the Applied Biosystems 3730 and 3730xl

DNA analyzers 48 and 96 capillary configuration 15 minute read-out (36 cm capillaries) 36cm Array & POP-7 used

Assay Chemistry: Triple Ligation

The SNPlexTM Assay

Utilizes:– 3 unique unlabeled ligation probes per SNP

locus– Universal linker sets in multiplex OLA– Two unlabeled universal PCR primers for

PCR amplification– Universal set of dye labeled mobility

modifiers (ZipChute™ probes) that are identified by CE

– Universal reagent kits

Basic Assay Steps

EncodingGeneration of genotype (GT) specific products

through multiplex oligonucleotide ligation reaction (OLA)

AmplificationMultiplex PCR with universal primers

DecodingHybridization of universal ZipChute probes to

amplicons and identification of eluted ZipChutes by CE

SNPlex™ Assay Workflow Overview

Hybridize ZipChute™ SetHybridize ZipChute™ Set

Capture toSA-coated plates

Capture toSA-coated plates

Wash, Elute & Load on 3730Wash, Elute & Load on 3730

Primary Analysis & QC dataPrimary Analysis & QC data

Prep genomic DNAPrep genomic DNA

Purify by enzymatic digestionPurify by enzymatic digestion

Universal PCRUniversal PCR

Perform OLAPerform OLA Allelic Discrimination

Ligation Product Amplification

Anneal Reporter Probe

Read Out on CE

Allele Calling

Start

Data~ 16 hrs

Kinase probes and linkersKinase probes and linkers Activate probes

Purification

Purification

comments

• We are working on DNA quality/quantity assessment, as well as whole genome amplification quantitation

• Quantitation : Real Time PCR preferred.

comments

• Separation of pre and post-PCR area is a key point

Wash and eluteDrag Chutes Load on CE instrument

Detection on CE

SNP 1

SNP 21 run : 15 min = 4608 Data points

Function of ZipChute Probes

• The same Universal ZipChute mix is used for hybridization to each multiplex reaction.

• ZipChute probes that specifically hybridized to genotype specific single-stranded amplicons are eluted and analyzed by CE.

ZipChute identification is used for genotype determination.

Universal ZipChute™ Probes Offer Reproducible, High Throughput Detection

Migration des ZipChutes = Allelic Ladder

Sample Data

Marker (SNP)Allele 1 (bin)Allele 2 (bin

Allele 1 called

GeneMapper Software Visualization Tools Simplifies Data QC

an Applera Corporation Business

Cartesian PolarSample View

Genotype clustering

Polar Plot Clustering

Quality Values Enable Rapid Batch Analysis

Probe Ordering/myScience Interface

Unrestricted access since June 21st

SNPlex Assay Design and Ordering Process1. myScience login

• SNPlex submission will be open to all users

2. SNP submission• rs #

• hCV #

• FASTA

3. Format check• Checks for valid ID or file format

• Determines if content is sufficient for assay design

SNPlex Assay Design and Ordering Process4. Assay design submission

• Format-checked SNPs are submitted for multiplex pool design

• User selects organism and pooling strategy

5. Review design results• List of multiplex pools and SNPs in each pool

• List of SNPs not included in pools and reason

6. Order assays• Customer adds assays to shopping basket

myScience Environment Supports Multiple Assay Inputs

Pipeline Confirms Assay Format

User Defined Parameters Ensure Customized Content

Max number of SNPs Or

Minimum number of panels

Design Report Provides Comprehensive Summary

Why these 63 were not included?

Design Pipeline

• Genome Screen (BLAST)– Only for human targets

– Checks for uniqueness of sequence

• Assay Rules (probe design)– Checks for poly-weak bases (A/T – more than 9 in a row)

– Checks for poly-G (more than 5 in a row)

• Pooling Rules– Looks for cross-reactivity between assay components (probes,

ZipChutes, linkers, DNA)

• Small Multiplex– Not enough SNPs to manufacture an additional multiplex pool

– Current limit is 24

Should “failed” SNPs be resubmitteed?• Genome Screen: NO

– If it’s not unique today, it won’t be unique tomorrow

• Assay Rules: NO– Probe design will not change unless the design pipeline

changes

• Pooling Rules: YES– Some SNPs may work in larger submissions or in

combination with other SNPs

• Small Multiplex: YES– These SNPs are good! There were just too few of them.

What do you need?

• A 3730xl• A dedicated capillary array• GeneMapper v3.5

• 2 separates rooms• Appropiate robotics and thermocyclers

• A probe set submitted• A starter kit : all included except probes