1 / 36

SNPlex ™ Genotyping System: A New High Throughput Solution

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

javan
Download Presentation

SNPlex ™ Genotyping System: A New High Throughput Solution

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. SNPlex™ Genotyping System:A New High Throughput Solution

  2. 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

  3. Assay Chemistry: Triple Ligation

  4. 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

  5. Amplification Multiplex PCR with universal primers Decoding Hybridization of universal ZipChute probes to amplicons and identification of eluted ZipChutes by CE Basic Assay Steps Encoding Generation of genotype (GT) specific products through multiplex oligonucleotide ligation reaction (OLA)

  6. SNPlex™ Assay Workflow Overview Start Prep genomic DNA Kinase probes and linkers Activate probes Perform OLA Allelic Discrimination Purify by enzymatic digestion Purification Universal PCR Ligation Product Amplification Capture to SA-coated plates Purification Hybridize ZipChute™ Set Anneal Reporter Probe Wash, Elute & Load on 3730 Read Out on CE Data ~ 16 hrs Primary Analysis & QC data Allele Calling

  7. comments • We are working on DNA quality/quantity assessment, as well as whole genome amplification quantitation • Quantitation : Real Time PCR preferred.

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

  9. Detection on CE Wash and elute Drag Chutes Load on CE instrument SNP 1 SNP 2 1 run : 15 min = 4608 Data points

  10. 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.

  11. Universal ZipChute™ Probes Offer Reproducible, High Throughput Detection

  12. Migration des ZipChutes = Allelic Ladder

  13. Allele 1 (bin) Allele 2 (bin Marker (SNP) Allele 1 called Sample Data

  14. an Applera Corporation Business GeneMapper Software Visualization Tools Simplifies Data QC Sample View Cartesian Polar

  15. Genotype clustering

  16. Polar Plot Clustering

  17. Quality Values Enable Rapid Batch Analysis

  18. Probe Ordering/myScience Interface Unrestricted access since June 21st

  19. SNPlex Assay Design and Ordering Process • myScience login • SNPlex submission will be open to all users • SNP submission • rs # • hCV # • FASTA • Format check • Checks for valid ID or file format • Determines if content is sufficient for assay design

  20. SNPlex Assay Design and Ordering Process • Assay design submission • Format-checked SNPs are submitted for multiplex pool design • User selects organism and pooling strategy • Review design results • List of multiplex pools and SNPs in each pool • List of SNPs not included in pools and reason • Order assays • Customer adds assays to shopping basket

  21. myScience Environment Supports Multiple Assay Inputs

  22. Pipeline Confirms Assay Format

  23. User Defined Parameters Ensure Customized Content Max number of SNPs Or Minimum number of panels

  24. Design Report Provides Comprehensive Summary Why these 63 were not included?

  25. 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

  26. 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.

  27. 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

More Related