Development of a high-throughput high-density SNP genotyping array for bovine
SNPlex ™ Genotyping System: A New High Throughput Solution
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Transcript of SNPlex ™ Genotyping System: A New High Throughput Solution
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SNPlex™ Genotyping System:A New High Throughput Solution
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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
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Assay Chemistry: Triple Ligation
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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
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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
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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
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comments
• We are working on DNA quality/quantity assessment, as well as whole genome amplification quantitation
• Quantitation : Real Time PCR preferred.
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comments
• Separation of pre and post-PCR area is a key point
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Wash and eluteDrag Chutes Load on CE instrument
Detection on CE
SNP 1
SNP 21 run : 15 min = 4608 Data points
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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.
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Universal ZipChute™ Probes Offer Reproducible, High Throughput Detection
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Migration des ZipChutes = Allelic Ladder
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Sample Data
Marker (SNP)Allele 1 (bin)Allele 2 (bin
Allele 1 called
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GeneMapper Software Visualization Tools Simplifies Data QC
an Applera Corporation Business
Cartesian PolarSample View
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Genotype clustering
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Polar Plot Clustering
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Quality Values Enable Rapid Batch Analysis
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Probe Ordering/myScience Interface
Unrestricted access since June 21st
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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
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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
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myScience Environment Supports Multiple Assay Inputs
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Pipeline Confirms Assay Format
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User Defined Parameters Ensure Customized Content
Max number of SNPs Or
Minimum number of panels
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Design Report Provides Comprehensive Summary
Why these 63 were not included?
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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
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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.
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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