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Sponsored by Horizon Diagnostics: 
The Use of Standards in NGS: Managing Variability in NGS? 
Dr Sarah Leigh Brown, Cambri...
Disclaimer 
This Presentation does not constitute or form any part of an offer to sell, or invitation to purchase or apply...
33 
Genome Translation 
precision genome editing 
(GENESIS™) 
Modelling patient genetic variations 
that define disease bi...
NGS Labs 
• Support workflow 
analysis and setup 
• Provision of positive 
reference standard for 
everyday run control 
M...
NGS Workflow and sources of variability 
Tumour sample 
Action 
Analysis 
DNA extraction 
Variant Calling/ 
Confidence Sco...
Overview of HDx™ Reference Standards 
Product Product Detail Features Application 
Genomic DNA Singleplex Defined allelic ...
Creating cell lines- Horizon Discovery’s Gene Editing Platform 
7 
“Wild type cell line” 
Single Cell Dilute 
Clonal wild ...
Cell Validation Test Assay 
Confirm identity of parental cell line STR and/or SNP6.0 
Confirm integration in the correct 
...
Genomic DNA Standards verified using Digital™ PCR 
Gene Mutation Allelic Frequency 
BRAF V600K 50% 10% 5% 1% 0.5% 0.1% 0.0...
Validation of NGS somatic variant pipelines 
Parameters that may be assessed: 
• Sensitivity do you detect the targets if...
Multiplex Reference Standards for NGS workflow analysis 
Genetically Defined Mutant Cell Lines 
Precise DNA dilutions 
Dig...
Case Study Data for Q-Seq FFPE HDx™ Reference Standards 
Source: 
Horizon 
Discovery 
Partner A Partner B Partner C Partne...
Q-Seq HDx™ Reference Standards for Next Generation Sequencing 
EGFR 
mutants 
K-Ras 
mutants 
B-Raf 
mutants 
N-Ras 
mutan...
Case Study Data for Tru-Q HDx™ Reference Standards 
Source: 
Horizon 
Diagnostics 
Predicted % 
5% blend 2.5% blend 1.3% b...
 Horizon Diagnostics’ suite of reference material includes standards for the increasing number of ‘rare’ 
mutations being...
What’s coming next? 
Low Copy Number / 
cfDNA Reference Standards 
Translocation / RNA 
Reference Standards 
…and more
Your Horizon Contact: 
Louisa Ludbrook 
Head of Sales 
l.ludbrook@horizondiscovery.com 
+44 1223 655589 
www.horizondx.com...
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HDx™ Reference Standards and Reference Materials for Next Generation Sequencing somatic variant pipelines

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HDx™ Reference Standards and Reference Materials for Next Generation Sequencing somatic variant pipelines

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HDx™ Reference Standards and Reference Materials for Next Generation Sequencing somatic variant pipelines

  1. 1. Sponsored by Horizon Diagnostics: The Use of Standards in NGS: Managing Variability in NGS? Dr Sarah Leigh Brown, Cambridge Institute Genomics Core, CRUK-CI. HDx™ Reference Standards and Reference Materials for Next Generation Sequencing somatic variant pipelines Dr Louisa Ludbrook, Horizon Discovery, Cambridge, UK NGS Sheffield, Nov 11th 2014
  2. 2. Disclaimer This Presentation does not constitute or form any part of an offer to sell, or invitation to purchase or apply for or enter into any contract or make any other commitment whatsoever in relation to, securities. Although reasonable care has been taken to ensure that the facts stated in this Presentation are accurate and that the opinions expressed are fair and reasonable, the contents of this Presentation have not been formally verified by Horizon Discovery plc (the “Company”) or any other person. Accordingly, no representation or warranty, expressed or implied, is made as to the fairness, accuracy, completeness or correctness of the information and opinions contained in this Presentation and no reliance should be placed on such information or opinions. Further, the information in this Presentation is not complete and is subject to updating, revision, further verification and amendment. Neither the Company, nor any of its subsidiaries, nor any of its respective members, directors, officers or employees nor any other person accepts any liability whatsoever for any loss howsoever arising from any use of such information or opinions or otherwise arising in connection with this Presentation. Accordingly, information contained in the Presentation is being supplied to you solely for your information and may not be copied, reproduced or further distributed to any person or published in whole or in part, for any purpose. In particular, the distribution of this Presentation in certain jurisdictions may be restricted by law, and persons into whose possession this Presentation comes should inform themselves about, and observe, any such restrictions. Any failure to comply with these restrictions may constitute a violation of laws of any such jurisdiction. This Presentation includes certain forward-looking statements, estimates and projections with respect to the anticipated future performance of Horizon Discovery plc, its products and the markets in which it operates. Forward-looking statements involve risks and uncertainties. Actual events could differ materially from those projected herein and such statements, estimates and projections reflect the various assumptions made by the Company which assumptions may or may not prove to be correct. These forward-looking statements speak only as at the date of this Presentation. The Company expressly disclaims any obligation or undertaking to disseminate any updates or revisions to any forward-looking statements contained in the Presentation to reflect any change in the Company’s expectations with regard thereto or any change in events, conditions or circumstances on which any such statements are based. No part of this Presentation, or the fact of its distribution, should form the basis of or be relied upon in connection with any contract or commitment or investment decision whatsoever. This Presentation does not constitute a recommendation regarding the securities of the Company. By participating in and/or accepting delivery of this Presentation you agree to be bound by the foregoing restrictions and the other terms of this disclaimer.
  3. 3. 33 Genome Translation precision genome editing (GENESIS™) Modelling patient genetic variations that define disease biology, drug response & resistance Precision functional genomics; targeted gene knock-outs & knock-ins Capacity to make 200 models / year Custom generation services Human Cellular Disease Models Gene-X, Mutant And Normal isogenic cell lines X-MAN™ Genetically defined disease models & endogenous pathway reporters to study target biology Rational screening tools for novel biomarker & target-ID & validation Drug screening >500 off-the-shelf disease models Tumour Microenvironment Assay development & screening CRO (Horizon Discovery Services) Suite of specialist assays including hypoxia, 3D, senescence, autophagy & pathway analysis Endogenous mutations placed under selective pressure to illicit true disease phenotypes & drug responses Portfolio of 50+ 2D and 3D Assays Right Drug Right Patient X-MAN™ surrogate patient profiling Predicting responsive or resistant patients before entering clinical trials using wide panels of X-MAN disease models Drug profiling service COLTHERES, PREDICT, INSIGHT EU FP6 Translational Consortia HDx™ Reference Standards Genetically defined DNA or paraffin embedded cells in specific wild type vs. mutant ratios Portfolio of genomic reference standards for multiple platforms; Sanger, qPCR, NGS, IHC & FISH What we do
  4. 4. NGS Labs • Support workflow analysis and setup • Provision of positive reference standard for everyday run control Molecular Labs • Support bridging studies and setup of NGS • Provision reference standards for singleplex assays OEM Partnerships • Support assay/platform development • Provision of reference material for validation studies • Supply of internal kit reference standards HDx™ Reference Standards Every Assay, Every Run, Every Confidence
  5. 5. NGS Workflow and sources of variability Tumour sample Action Analysis DNA extraction Variant Calling/ Confidence Scoring Reference Materials DNA Quantification Library Preparation Sequencing Alignment/Mapping
  6. 6. Overview of HDx™ Reference Standards Product Product Detail Features Application Genomic DNA Singleplex Defined allelic frequency (e.g. KRAS 1% G12A) 6 Routinely monitor the performance of your molecular assay (Sanger Sequencing, Therascreen,…) Multiplex Multiple mutations in one tube (e.g. EGFR 5% G719S; 5% L858R, 5% T790M,…) Monitor the performance of your NGS platform FFPE Sections Singleplex or Multiplex Defined standards in FFPE format Monitor the workflow from DNA extraction to genotyping FFPE Blocks Standard Genotype A complete block (~800sections@15μm) Consistent material for assay validation studies Multiplex Genotype Select your genotypes Select your allelic frequencies Consistent and customised material for assay validation studies Cell Slides FISH Slides Defined chromosomal abnormalities (EML/ALK) Validate your FISH probes and workflow IHC Slides COMING SOON Defined protein expression levels (ROS1, c-MET, EGFR, HER2, EML4/ALK) Routinely monitor the performance of IHC workflow
  7. 7. Creating cell lines- Horizon Discovery’s Gene Editing Platform 7 “Wild type cell line” Single Cell Dilute Clonal wild type cell line Cell Line Validation Gene Engineering Technology (GENESIS™) Clonal mutant cell line Cell Line Validation
  8. 8. Cell Validation Test Assay Confirm identity of parental cell line STR and/or SNP6.0 Confirm integration in the correct locus 8 Cell Line Validation Clonal heterozygous mutant cell line gDNA locus specific PCR & Sanger Sequencing Confirm expression of modified allele cDNA-PCR & Sanger Sequencing Confirm clonality Droplet digital PCR, gDNA PCR & Sanger Sequencing Confirm gene copy number Droplet digital PCR Clonal wild type cell line Fully validated genetically defined isogenic cell line pair
  9. 9. Genomic DNA Standards verified using Digital™ PCR Gene Mutation Allelic Frequency BRAF V600K 50% 10% 5% 1% 0.5% 0.1% 0.05% KRAS G12D 50% 10% 5% 1% 0.5% 0.1% 0.05% Stoichiometric dilutions are accurate down to 0.1% and 0.05%
  10. 10. Validation of NGS somatic variant pipelines Parameters that may be assessed: • Sensitivity do you detect the targets if they are present? • Specificity can you distinguish between false/true positives? • Limit of Detection what AF is your limit, does it vary mutation to mutation? • Repeatability run-to-run, same and different variants • Reproducibility inter-lab, same result elsewhere? • Robustness what is the effect of sample quality on performance? DNA and FFPE HDx™ Reference Standards are manufactured within a Quality Management System which complies with the requirements of ISO 9001:2008 and ISO 13485:2003.
  11. 11. Multiplex Reference Standards for NGS workflow analysis Genetically Defined Mutant Cell Lines Precise DNA dilutions Digital PCR Analysis Quantitative Multiplex Reference Standards  Quantitative Multiplex Reference Standard  A ‘ladder’ of 11 engineered fixed allelic frequency variants (1% to 24.5% AF)  Validated endogenous variants (20)  Predicted endogenous variants (>200)  Genomic DNA Format: 1ug @ 50ng/μl - £160  FFPE Format: Vial containing 1 FFPE section that will provide >400ng (5 sections - £195)  Formalin Compromised (Paraffin-Free) DNA Grade I and Grade II formalin intensity (1ug - £160)
  12. 12. Case Study Data for Q-Seq FFPE HDx™ Reference Standards Source: Horizon Discovery Partner A Partner B Partner C Partner D Platform: QX100™ Droplet Digital™ PCR System AmpliSeq Cancer Panel Ampliseq Cancer Hotspot Panel v2 Ampliseq Cancer Hotspot Panel v2 (Average of 8 runs) TruSeq Panel Sequencing Depth N/A 3000-4000x Average 5000x 2000X 150k+ Gene Mutation Observed mutant ratio BRAF V600E 10.2 9.9 9.1 10.3 9 KIT D816V 10.4 10.0 11.0 10.1 Not Tested EGFR ΔE746 - A750 2.0 2.3 Not detected Not detected Not Tested EGFR L858R 2.7 2.7 2.1 2.4 Not Tested EGFR T790M 0.9 0.8 Not detected Not detected Not Tested EGFR G719S 24.4 23.7 23.1 24.8 Not Tested KRAS G13D 16.1 16.3 12.35 15.5 15.4 KRAS G12D 5.0 5.2 Not detected 5.1 5.5 NRAS Q61K 12.8 9.0 12.7 12.6 13.5 PIK3CA H1047R 18.6 16.7 16.8 17.9 17.0 PIK3CA E545K 8.9 3.2 8.4 8.8 10.0 Multiplex Reference Standard can be used to identify false negative NGS reads
  13. 13. Q-Seq HDx™ Reference Standards for Next Generation Sequencing EGFR mutants K-Ras mutants B-Raf mutants N-Ras mutants PIKCA mutants Quantification by Droplet Digital PCR C Blend 1 10 mutations at 5% C Blend 2 10 mutations at 5% C Blend 3 10 mutations at 5% Quantification by Droplet Digital PCR Quantification by Droplet Digital PCR A Blend 40 Mutations @ 1.3% B Blend 1 20 Mutations at 2.5% B Blend 2 20 Mutations at 2.5% C Blend 4 10 mutations at 5% 14 Additonal Biomarkers 1.3% 20 copies per μl Quantification by Droplet Digital PCR
  14. 14. Case Study Data for Tru-Q HDx™ Reference Standards Source: Horizon Diagnostics Predicted % 5% blend 2.5% blend 1.3% blend Horizon Diagnostics Observed % Partner Platform: N/A QX100™ Droplet Digital™ PCR System Ion Torrent Gene Mutatio n BRAF V600M 4.0 4.4 3.5 EGFR T790M 4.2 3.9 4.3 EGFR L858R 4.2 4.2 3.5 EGFR L861Q 4.2 4.1 3.6 KIT D816V 5.0 5.4 6.4 KRAS G12A 5.0 5.7 4.9 KRAS G12R 5.0 5.2 4.6 NRAS Q61K 5.0 4.9 3.3 Specific and Sensitive down to 5% allelic frequency Horizon Diagnostics Predicted % Horizon Diagnostics Observed % Partner N/A QX100™ Droplet Digital™ PCR System Ion Torrent 2.0 2.2 2.1 2.1 2.0 2.1 2.1 2.0 2.3 2.1 2.1 1.8 2.5 2.6 3.2 2.5 3.0 2.5 2.5 2.9 2.6 2.5 2.5 2.5 Horizon Diagnostics Predicted % Horizon Diagnostics Observed % Partner N/A QX100™ Droplet Digital™ PCR System Ion Torrent 1.0 1.0 1.9 1.0 1.1 missing 1.0 1.1 missing 1.0 1.0 missing 1.3 1.3 1.5 1.3 1.4 missing 1.3 1.3 missing 1.3 1.2 missing Specific and Sensitive down to 2.5% allelic frequency Not sensitive to detect down to 1% for all variants
  15. 15.  Horizon Diagnostics’ suite of reference material includes standards for the increasing number of ‘rare’ mutations being targeted for cancer therapeutics, which by definition are hard to find in clinical samples. ABL1 AKT1 ALK BRAF cKIT EGFR FGFR2 FLT3 GNAQ GNA11 IDH1 IDH2 JAK2 KRAS G12V E17K Q209L V600E V600K V600R R132C R132H G719S T790M L858R L861Q ΔE746-A750 V617F S252W G12A G12C G12D G12R G12S G12V G13D T315I D835Y L1601P F1174L R1275Q F1245V Q209L Q61H Q61K Q61L Q61R D816V R140Q R172K E542K E545K H1047R EML4/ALK V600M V600G Δ1836 NOTCH1 MET NRAS MLL PI3K Y1253D MLL/ENL PTEN ΔEX6/EX7 ROS1 ROS1 RUNX1 RUNX1/RUNX1T1 Q61H A146T Mutation Coverage
  16. 16. What’s coming next? Low Copy Number / cfDNA Reference Standards Translocation / RNA Reference Standards …and more
  17. 17. Your Horizon Contact: Louisa Ludbrook Head of Sales l.ludbrook@horizondiscovery.com +44 1223 655589 www.horizondx.com Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom Tel: +44 (0) 1223 655 580 (Reception / Front desk) Fax: +44 (0) 1223 655 581 Email: info@horizondx.com Web: www.horizondx.com

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