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
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
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
DNA Sequencing: Interrogating The 1° Structure
Unknown
Patient DNA
Sequence
3’ – X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
5’ – X
Chain Termination
AKA: Sanger Sequencing
Known
Patient DNA
Sequence
A
T
G
C
T
T
C
G
G
C
A
A
G
A
C
T
C
A
A
A
A
A
A
T
A
Align
&
Compare
“Call’ the
Variant
“Read”
length ~700 bases
Reference
DNA
Sequence
C – 3’
T
A
T
G
C
T
T
C
G
G
C
A
T
G
A
C
T
C
A
A
A
A
A
A
T
A
C
C
G – 5’
Assign Variant Annotation
&
Interpret for Clinic
DNA Sequencing: Genomic Variation
Known
Patient DNA
Sequence
A
T
G
C
T
T
C
G
G
C
A
A
G
A
C
T
C
A
A
A
A
A
A
T
A
Align
&
Compare
“Call’ the
Variant
“Read”
length ~700 bases
Reference
DNA
Sequence
C – 3’
T
A
T
G
C
T
T
C
G
G
C
A
T
G
A
C
T
C
A
A
A
A
A
A
T
A
C
C
G – 5’
Genomic Variation = Different from Reference
Genomic variants are either inherited or de novo
• Single Nucleotide Polymorphism (SNP)
• Tandem Repeats (STR, microsatellite)
• Insertion
• Deletion
• Amplification
• Inversion
• Translocation
• Aneuploidy
Pathogenic?
Next Generation Sequencing
Next Generation = Massively Parallel
PATIENT 5’- XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX - 3’
“Read” TGAACTAGTCTCGGA
length 35-300 bases
CGATGAACTAGTCTC
CTCGGACCCGTAATG
“Read Depth”
TTTCGATGAACTAGT
“Depth of Coverage”
GACCCGTAATGGTCT
ATTTCGATGAACTAG
“Coverage”
CTAGTCTCGGACCCG
GTGCCATTTCGATGA
“X-Fold Coverage”
TCGATGAACTAGTCT
ACTAGTCTCGGACCC
GATGAACTAGTCTCG
CCATTTCGATGAACT
REFERENCE 5’- AGTGCCATTTCGATGAACATGTCTCGGACCCGTAATGGTCTCTTGGGTCTGAA - 3’
False Positive
TAT
Correct
False Negative
Call Rate
&
Accuracy
Next Next Generation Sequencing
Next Next Generation = Massively Parallel Massively Parallel
Sanger
Next Generation
Highly Centralized Sequencing Factories
-orHighly Distributed Desktop Sequencers
Next Next Generation
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
Next Next Gen
Next Gen
Microarray
Sanger
DNA Sequencing: Approaches to the Genome
Process of Clinical Genome Sequencing
PATIENT
Results
Delivery
Sample
Acquisition
Report
Generation
Accessioning
Genomic
DNA
Isolation
Clinical
Interpretation
Data
Packaging
& Delivery
gDNA
Quality
Control
Variant
Annotation
Library
Construction
Variant
Calling
Sequencing
Assembly
Imaging
Economics of Genome Sequencing
Cost Per Genome
$2MM
100,000
2003: $2,300,000,000 / 15 years
2014: $2,300 / 15 days
20K
$350K
Number of Whole Genomes Sequenced
1989
1
2
2003
2007
9
$5K
2011
$2.3K
2014
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
Moving WGS to the Clinic
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
WGS: Proving Clinical Utility
“A test that is analytically sound but has no
established clinical utility should not be offered
Jennings, et al. Recommended Principles & Practices for Validating
clinically.”
Clinical Molecular Pathology Tests. Arch Pathol Lab Med. V133,
May 2009
“…expresses- preferably in a quantitative formto what extent diagnostic testing improves
health outcomes relative to the current best
et al. Beyond Clinical Diagnostic Accuracy: The Clinical
alternative.” Bossuyt,
Utility of Diagnostic Tests. Clinical Chemistry- V58, December 2012
Clinical Utility Study Program
• 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
WGS: Current Clinical Utility Studies
18 collaborative studies ongoing
• Cardiology
• Congenital Malformation
• Developmental Delay / Intellectual Disability
• Health Economics
• Neurology
• Newborn Screening
• Oncology
• Ophthalmology
• Pathology
Clinical Utility Study Program
Oncology: Oxford Weatherall Institute
• Prof. Ahmed Ashour Ahmed
UK
• 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
*Peters & Drmanac, et al. Nature, Vol. 487, July 2012
Epilepsy: UCL / NHS
UK
• Large Epilepsy study – hundreds of genomes
• Brain surgery is current standard of care for
certain debilitating epilepsies
• Utility of WGS for surgical outcome prediction
ASD / DDID / Malformation
Canada
• 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
Intellectual Disability: RUNMC
• 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
2012: Diagnostic Exome 100 ID Trios
All negative by Sanger, Microarray
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
16% Diagnostic Yield
3
Autosomal Dominant
0
Autosomal Recessive
0
X-Linked
3
Candidate Causal Variants
19
No Diagnosis
65
De Ligt, et al. New Engl J Med. Vol 367, November 2012
Netherlands
2013: Whole Genome 50 ID trios
All negative by Sanger, Microarray, Exome
POSITIVE 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
Netherlands
Evolution of Diagnostic Yield for ID
Netherlands
2005
Karyotyping
Karyotyping
Sanger Sequencing
Sanger Sequencing
Clinical Microarray
Clinical Microarray
Exome Sequencing
Karyotyping
Exome Sequencing
Genome Sequencing
Sanger Sequencing
Genome Sequencing
No Diagnosis
Clinical Microarray
No Diagnosis
2010
Exome Sequencing
Genome Sequencing
No Diagnosis
2013
Karyotyping
Karyotyping
Sanger Sequencing
Sanger Sequencing
Clinical Microarray
Clinical Microarray
Exome Sequencing
2015
Exome Sequencing
Genome Sequencing
Genome Sequencing
No Diagnosis
No Diagnosis
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
Why do WGS for the clinic?
To extend and improve the quality of human life.
PERSONALIZED MEDICINE / PRECISION MEDICINE
• Treatment focused
• Uses panomics to select the right treatment for the right person at the right time.
• Reactive Medicine
P4 MEDICINE
• Wellness focused
• Personalized
• Preventive
• Predictive
• Participatory
• Proactive Medicine
Pharmacogenomics
•
•
•
•
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•
Good Drug
Antiplatelet
CYP2C19 variant
>15% non-metabolizers
FDA black box warning
~$1BB wasted per year
Pharmacogenomics
abacavir
acenocoumarol
acetaminophen
allopurinol
amitriptyline
aripiprazole
arsenic trioxide
atomoxetine
atorvastatin
azathioprine
boceprevir
brentuximab vedotin
capecitabine
carbamazepine
carisoprodol
carvedilol
celecoxib
cetuximab
cevimeline
chloroquine
cisplatin
citalopram
clobazam
clomifene
clomipramine
clopidogrel
clozapine
codeine
crizotinib
dapsone
dasatinib
denileukin diftitox
desipramine
dextromethorphan
diazepam
doxepin
drospirenone
duloxetine
eltrombopag
erlotinib
escitalopram
esomeprazole
ethinyl estradiol
everolimus
exemestane
flecainide
fluorouracil
fluoxetine
flurbiprofen
fluvoxamine
fulvestrant
galantamine
gefitinib
glibenclamide
gliclazide
glimepiride
haloperidol
hormonal contraceptives
hydralazine
iloperidone
imatinib
imipramine
indacaterol
irinotecan
isoniazid
isosorbide dinitrate
ivacaftor
lansoprazole
lapatinib
letrozole
maraviroc
mercaptopurine
methylene blue
metoprolol
mirtazapine
moclobemide
modafinil
mycophenolic acid
nalidixic acid
nelfinavir
nilotinib
nitrofurantoin
norfloxacin
nortriptyline
olanzapine
omeprazole
oxycodone
panitumumab
pantoprazole
paroxetine
peginterferon alfa-2b
pegloticase
perphenazine
Pertuzumab
phenprocoumon
phenylacetic acid
phenytoin
pimozide
prasugrel
pravastatin
primaquine
probenecid
propafenone
propranolol
protriptyline
pyrazinamide
quinidine
rabeprazole
rasburicase
ribavirin
rifampin
risperidone
sertraline
simvastatin
sodium benzoate
sulfadiazine
sulfamethoxazole
sulfasalazine
sulfisoxazole
tacrolimus
tamoxifen
tegafur
telaprevir
terbinafine
tetrabenazine
thioguanine
thioridazine
ticagrelor
timolol
tiotropium
tolbutamide
tolterodine
tositumomab
tramadol
trastuzumab
trastuzumab emtansine
tretinoin
trimethoprim
trimipramine
valproic acid
vemurafenib
venlafaxine
voriconazole
warfarin
zuclopenthixol
Parental Screening / Family Planning
Alpha-Thalassemia
Beta-Thalassemia
Bloom Syndrome
Canavan Disease
Cystic Fibrosis
Familial Dysautonomia
Current targeted
screening will be
replaced by whole
genome screening.
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
Pre-Symptomatic Diagnosis
Richard A. Leach, Ph.D.
Jeffrey Gulcher, M.D., Ph.D.
Idiopathic Disease Resolution
Nick Volker
Jacob
Mayer
Dimmock
Margolis
Verbsky
Worthey
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