Current Perspectives: Clinical Applications For Whole Genome Sequencing

Richard A. Leach, Ph.D.Vice President | Business Development | Complete Genomics

Conflict of Interest Disclosure

Vice President | Business Development

Board Member | Stakeholder

1. Understand the process of Whole Genome Sequencing (WGS) from tissue to data

2. Become familiar with relevant WGS quality metrics, genomic variants

3. Current perspective on clinical utility studies for WGS

4. Learn current and future clinical applications of WGS with some emphasis on preimplantation genetic diagnosis (PGD screening) and parental carrier screening

5. Clinical examples of WGS

CME Learning Objectives

1. Understand the process of Whole Genome Sequencing (WGS) from tissue to data

2. Become familiar with relevant WGS quality metrics, genomic variants

3. Current perspective on clinical utility studies for WGS

4. Learn current and future clinical applications of WGS with some emphasis on preimplantation genetic diagnosis (PGD screening) and parental carrier screening

5. Clinical examples of WGS

CME Learning Objectives

DNA Sequencing: Interrogating The 1° Structure

Assign Variant Annotation&

Interpret for Clinic

C – 3’TATGCTTCGGCATGACTCAAAAAATACCG – 5’

Align&

Compare

ReferenceDNASequence

“Call’ theVariant

Chain TerminationAKA: Sanger Sequencing

3’ – XXXXXXXXXXXXXXXXXXXXXXXXXXXXX

5’ – X

UnknownPatient DNASequence

ATGCTTCGGCAAGACTCAAAAAATA

KnownPatient DNASequence

“Read”length ~700 bases

DNA Sequencing: Genomic Variation

Genomic variants are either inherited or de novo

• Single Nucleotide Polymorphism (SNP)

• Tandem Repeats (STR, microsatellite)

• Insertion

• Deletion

• Amplification

• Inversion

• Translocation

• Aneuploidy

Genomic Variation = Different from ReferenceReferenceDNASequence

C – 3’TATGCTTCGGCATGACTCAAAAAATACCG – 5’

Align&

Compare

“Call’ theVariant

ATGCTTCGGCAAGACTCAAAAAATA

KnownPatient DNASequence

“Read”length ~700 bases

Pathogenic?

PATIENT 5’- XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX - 3’

Next Generation Sequencing

Next Generation = Massively Parallel

TGAACTAGTCTCGGA“Read”length 35-300 bases

REFERENCE 5’- AGTGCCATTTCGATGAACATGTCTCGGACCCGTAATGGTCTCTTGGGTCTGAA - 3’CCATTTCGATGAACT

GATGAACTAGTCTCGACTAGTCTCGGACCC

TCGATGAACTAGTCTGTGCCATTTCGATGA

CTAGTCTCGGACCCGATTTCGATGAACTAG

TTTCGATGAACTAGTGACCCGTAATGGTCT

CTCGGACCCGTAATGCGATGAACTAGTCTC

TATFalse Positive False Negative

Correct

“Read Depth”“Depth of Coverage”“Coverage”“X-Fold Coverage”

Call Rate&

Accuracy

Next Next Generation Sequencing

Next Next Generation = Massively Parallel Massively Parallel

Next GenerationSanger Next Next Generation

Highly Centralized Sequencing Factories-or-

Highly Distributed Desktop Sequencers

DNA Sequencing: Approaches to the Genome

Genotyping: Known SNP’s & Microsatellites

Targeted: Known finite regions

Whole Exome: Coding region (~1% of total)• Targeted Exome Capture (~180,000 exons)• No non-coding DNA (i.e. no introns, regulatory regions, etc.)

Whole Genome: Everything

San

ger

Nex

t G

en

Nex

t N

ext

Gen

Mic

roar

ray

PATIENTSample

Acquisition

Accessioning

Genomic DNA

Isolation

gDNA Quality Control

Library Construction

Sequencing

ImagingAssembly

Variant Calling

Variant Annotation

Data Packaging & Delivery

Clinical Interpretation

Report Generation

Results Delivery

Process of Clinical Genome Sequencing

Economics of Genome Sequencing

1989 2003 20142007 2011

2014: $2,300 / 15 days

100,000

$2.3K

20K

$5K9

$350K

Cost Per Genome $2MM

2

2003: $2,300,000,000 / 15 years

Number of Whole Genomes Sequenced 1

1. Understand the process of Whole Genome Sequencing (WGS) from tissue to data

2. Become familiar with relevant WGS quality metrics, genomic variants

3. Current perspective on clinical utility studies for WGS

4. Learn current and future clinical applications of WGS with some emphasis on preimplantation genetic diagnosis (PGD screening) and parental carrier screening

5. Clinical examples of WGS

CME Learning Objectives

Many interwoven and complicated challenges for clinical adoption of WGS as standard of care

WGS: Basic milestones for clinical adoption• Platform Validity• Proof of Clinical Utility• Health Economic Benefit

Moving WGS to the Clinic

“A test that is analytically sound but has no established clinical utility should not be offered clinically.” Jennings, et al. Recommended Principles & Practices for

Validating Clinical Molecular Pathology Tests. Arch Pathol Lab Med. V133, May 2009

“…expresses- preferably in a quantitative form- to what extent diagnostic testing improves health outcomes relative to the current best alternative.” Bossuyt, et al. Beyond Clinical Diagnostic Accuracy: The Clinical

Utility of Diagnostic Tests. Clinical Chemistry- V58, December 2012

WGS: Proving Clinical Utility

• Global program of basic collaborative studies

• Results published in leading clinical journals

• 1° Goal: Compare diagnostic yield of WGS versus existing standard of care

• 2° Goal: Demonstrate applications of WGS

• 2° Goal: Study Health Economic Benefit

Clinical Utility Study Program

18 collaborative studies ongoing• Cardiology• Congenital Malformation• Developmental Delay / Intellectual Disability• Health Economics• Neurology• Newborn Screening• Oncology• Ophthalmology• Pathology

WGS: Current Clinical Utility Studies

Clinical Utility Study Program

• Prof. Ahmed Ashour Ahmed

• Intra-operative monitoring of High Grade Serous Ovarian Cancer

• Multiple fine-needle biopsies from a single tumor before and after chemotherapy

• Used CGI Long Fragment Read Technology*

• Study completed, manuscript submitted

UK

*Peters & Drmanac, et al. Nature, Vol. 487, July 2012

Oncology: Oxford Weatherall Institute

• Large Epilepsy study – hundreds of genomes

• Brain surgery is current standard of care for certain debilitating epilepsies

• Utility of WGS for surgical outcome prediction

UK

Epilepsy: UCL / NHS

• Diagnostic yield of WGS vs. clinical microarray

• Prospective study: clinical assay performed in parallel with WGS

• Assessing frequency of medically actionable variants unrelated to 1° reason for testing

• Initial results very exciting

Canada

ASD / DDID / Malformation

• Prof. Han Brunner, Prof. Joris Veltman

• Severe ID usually due to de novo variation

• Diagnostic Yield of WGS vs. exome

• Previous exome study published in NEJM

• Current study 50 exome negative trios

• Variants in exome & regulatory regions

• Focus on de novo events

Netherlands

Intellectual Disability: RUNMC

2012: Diagnostic Exome 100 ID TriosAll negative by Sanger, Microarray

Netherlands

De Ligt, et al. New Engl J Med. Vol 367, November 2012

POSITIVE DIAGNOSIS NUMBER OF PATIENTS (n=100)

All Mutations 16

De Novo Mutations 13

Autosomal Dominant 10

Autosomal Recessive 1

X-Linked 2

Inherited Mutations 3

Autosomal Dominant 0

Autosomal Recessive 0

X-Linked 3

Candidate Causal Variants 19

No Diagnosis 65

16% Diagnostic Yield

2013: Whole Genome 50 ID triosAll negative by Sanger, Microarray, Exome

NetherlandsPOSITIVE DIAGNOSIS NUMBER OF PATIENTS (n=50)

All Mutations 19

De Novo Mutations 18

Autosomal Dominant 14

Autosomal Recessive 0

X-Linked 4

Inherited Mutations 1

Autosomal Dominant 0

Autosomal Recessive 1

X-Linked 0

Candidate Causal Variants 8

No Diagnosis 23

38% Diagnostic Yield

KaryotypingSanger SequencingClinical MicroarrayExome SequencingGenome SequencingNo Diagnosis

KaryotypingSanger SequencingClinical MicroarrayExome SequencingGenome SequencingNo Diagnosis

2005

KaryotypingSanger SequencingClinical MicroarrayExome SequencingGenome SequencingNo Diagnosis

2013

KaryotypingSanger SequencingClinical MicroarrayExome SequencingGenome SequencingNo Diagnosis

2015

KaryotypingSanger SequencingClinical MicroarrayExome SequencingGenome SequencingNo Diagnosis

2010

Netherlands

Evolution of Diagnostic Yield for ID

1. Understand the process of Whole Genome Sequencing (WGS) from tissue to data

2. Become familiar with relevant WGS quality metrics, genomic variants

3. Current perspective on clinical utility studies for WGS

4. Learn current and future clinical applications of WGS with some emphasis on preimplantation genetic diagnosis (PGD screening) and parental carrier screening

5. Clinical examples of WGS

CME Learning Objectives

P4 MEDICINE• Wellness focused• Personalized• Preventive• Predictive• Participatory

• Proactive Medicine

Why do WGS for the clinic?

PERSONALIZED MEDICINE / PRECISION MEDICINE• Treatment focused• Uses panomics to select the right treatment for the right person at the right time.

• Reactive Medicine

To extend and improve the quality of human life.

Pharmacogenomics• Good Drug• Antiplatelet• CYP2C19 variant• >15% non-metabolizers• FDA black box warning• ~$1BB wasted per year

abacavir clopidogrel fulvestrant moclobemide primaquine thioguanineacenocoumarol clozapine galantamine modafinil probenecid thioridazineacetaminophen codeine gefitinib mycophenolic acid propafenone ticagrelorallopurinol crizotinib glibenclamide nalidixic acid propranolol timololamitriptyline dapsone gliclazide nelfinavir protriptyline tiotropiumaripiprazole dasatinib glimepiride nilotinib pyrazinamide tolbutamidearsenic trioxide denileukin diftitox haloperidol nitrofurantoin quinidine tolterodineatomoxetine desipramine hormonal contraceptives norfloxacin rabeprazole tositumomabatorvastatin dextromethorphan hydralazine nortriptyline rasburicase tramadolazathioprine diazepam iloperidone olanzapine ribavirin trastuzumabboceprevir doxepin imatinib omeprazole rifampin trastuzumab emtansinebrentuximab vedotin drospirenone imipramine oxycodone risperidone tretinoincapecitabine duloxetine indacaterol panitumumab sertraline trimethoprimcarbamazepine eltrombopag irinotecan pantoprazole simvastatin trimipraminecarisoprodol erlotinib isoniazid paroxetine sodium benzoate valproic acidcarvedilol escitalopram isosorbide dinitrate peginterferon alfa-2b sulfadiazine vemurafenibcelecoxib esomeprazole ivacaftor pegloticase sulfamethoxazole venlafaxinecetuximab ethinyl estradiol lansoprazole perphenazine sulfasalazine voriconazolecevimeline everolimus lapatinib Pertuzumab sulfisoxazole warfarinchloroquine exemestane letrozole phenprocoumon tacrolimus zuclopenthixolcisplatin flecainide maraviroc phenylacetic acid tamoxifencitalopram fluorouracil mercaptopurine phenytoin tegafurclobazam fluoxetine methylene blue pimozide telaprevirclomifene flurbiprofen metoprolol prasugrel terbinafineclomipramine fluvoxamine mirtazapine pravastatin tetrabenazine

Pharmacogenomics

Parental Screening / Family PlanningAlpha-Thalassemia

Beta-Thalassemia

Bloom Syndrome

Canavan Disease

Cystic Fibrosis

Familial Dysautonomia

Familial Hyperinsulinism

Fanconi Anemia

Fragile X Syndrome

Gaucher Disease (Type I)

Glycogen Storage Disease 1A

Joubert Syndrome 2

Lipoamide Dehydrogenase Deficiency

Maple Syrup Urine Disease

Mucopolipidosis IV

Neiman Pick Type A

Nemaline Myopathy

Spinal Muscular Atrophy

Tay-Sachs Disease

Usher Syndrome

Walker-Warburg Syndrome

Current targeted screening will be replaced by whole genome screening.

Pre-Symptomatic Diagnosis

Richard A. Leach, Ph.D.

Jeffrey Gulcher, M.D., Ph.D.

Dimmock

Mayer

Jacob

Margolis

Verbsky

Worthey

Idiopathic Disease Resolution

Nick Volker

LFR: A Major Sequencing Advancement

LFR: A Major Sequencing Advancement

Long Fragment Read (LFR)• 10 cells starting material• <600 errors per diploid genome• Phased – 98%• Identify de novo variants• Parent of origin

Current Perspectives: Clinical Applications For Whole Genome Sequencing

Richard A. Leach, Ph.D.Vice President | Business Development | Complete Genomics