hdx™ reference standards and reference materials for next generation sequencing somatic variant...

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

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.

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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

Genome Translation

Human Cellular Disease ModelsGene-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 MicroenvironmentAssay 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 PatientX-MAN™ surrogatepatient 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

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 singleplexassays

OEM Partnerships• Support assay/platform

development• Provision of reference

material for validation studies

• Supply of internal kit reference standards

HDx™ Reference StandardsEvery Assay, Every Run, Every Confidence

NGS Workflow and sources of variability

Tumour sample

Analysis

Action

DNA extraction

DNA Quantification Library Preparation Sequencing Alignment/Mapping

Variant Calling/ Confidence Scoring

Reference Materials

6

Overview of HDx™ Reference Standards

Product Product Detail Features Application

Genomic DNA Singleplex Defined allelic frequency(e.g. KRAS 1% G12A)

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 SingleplexorMultiplex

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 genotypesSelect 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 SlidesCOMING SOON

Defined protein expression levels(ROS1, c-MET, EGFR, HER2, EML4/ALK)

Routinely monitor the performance of IHC workflow

7

Creating cell lines- Horizon Discovery’s Gene Editing Platform

“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

Cell Line Validation

Clonal heterozygousmutant cell line

Cell Validation Test Assay

Confirm identity of parental cell line STR and/or SNP6.0

Confirm integration in the correct locus

gDNA locus specific PCR & Sanger Sequencing

Confirm expression of modified allele cDNA-PCR & Sanger Sequencing

Confirm clonalityDroplet 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

Genomic DNA Standards verified using Digital™ PCR

Stoichiometric dilutions are accurate down to 0.1% and 0.05%

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%

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.

Multiplex Reference Standards for NGS workflow analysis

Precise DNA dilutions

Digital PCR Analysis

Quantitative Multiplex Reference Standards

Genetically Defined Mutant Cell Lines

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) DNAGrade I and Grade II formalin intensity (1ug - £160)

Multiplex Reference Standard can be used to identify false negative NGS reads

Source:Horizon

DiscoveryPartner A Partner B Partner C Partner D

Platform:QX100™ Droplet

Digital™ PCR System

AmpliSeq CancerPanel

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

EGFRΔE746 -

A7502.0 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

Case Study Data for Q-Seq FFPE HDx™ Reference Standards

EGFR mutants

K-Rasmutants

B-Rafmutants

N-Rasmutants

PIKCA mutants

Quantification by Droplet Digital PCR

C Blend 110 mutations

at 5%


at 5%


at 5%

A Blend40 Mutations

@ 1.3%

B Blend 120 Mutations

at 2.5%

B Blend 220 Mutations

at 2.5%


at 5%

14 AdditonalBiomarkers

1.3%20 copies per μl




Q-Seq HDx™ Reference Standards for Next Generation Sequencing

Source:Horizon

DiagnosticsPredicted %

Horizon DiagnosticsObserved %

Partner

Platform: N/A

QX100™ Droplet Digital™

PCR System

Ion Torrent

GeneMutatio

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

Case Study Data for Tru-Q HDx™ Reference Standards

Specific and Sensitive down

to 5% allelic frequency

Horizon DiagnosticsPredicted %


Partner

N/A


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 DiagnosticsPredicted %


Partner

N/A


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

5% blend 2.5% blend 1.3% blend

G12V

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.

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

ABL1 AKT1 ALK BRAF cKIT EGFR FGFR2

FLT3 GNAQ GNA11 IDH1 IDH2 JAK2 KRAS

NOTCH1 MET NRAS MLL PI3K

Y1253D MLL/ENL

PTEN

ΔEX6/EX7

ROS1

ROS1

RUNX1

RUNX1/RUNX1T1

Q61H

A146T

Mutation Coverage

What’s coming next?

Low Copy Number / cfDNA Reference Standards

Translocation / RNA Reference Standards

…and more

Your Horizon Contact:

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: [email protected] Web: www.horizondx.com

Louisa Ludbrook

Head of Sales

[email protected]

+44 1223 655589

www.horizondx.com

hdx™ reference standards and reference materials for next generation sequencing somatic variant...

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