investigating rare diseases with agilent ngs solutions rare diseases with agilent ngs solutions ......

Investigating rare diseases with Agilent NGS solutions

Chitra Kotwaliwale, Ph.D.

April 27, 2017

April 27, 2017

Research use only. Not for use in diagnostic purposes.

1

Rare diseases affect 350 million people worldwide

April 27, 2017


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60 million affected in

the US, Europe

50% affected individuals are

children

7,000 rare diseases

80% are genetic

Rare diseases can have devastating impact on health

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Cystic Fibrosis

• Excessive mucus in lungs and pancreas causes respiratory failure and inability to digest food

• Median survival age is 40 years

• Affects more than 30,000 people in the US; 70,000 WW

Leukodystrophy

• Progressive diseases that affect brain, spinal cord, peripheral nerves affecting movement, vision, hearing, balance, ability to eat etc.

• Children affected with leukodystrophy live 5-10 years

• Affects ~60,000 people in the US

Retinitis Pigmentosa

• Retinal degeneration ultimately causes blindness

• Most people with RP are legally blind by age 40

• Affects ~100,000 people in the US; 1.5 million WW

Genetic causes of rare diseases can be complex

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Cystic Fibrosis

1 gene

Leukodystrophy

30 genes

Retinitis

Pigmentosa

77 genes

Wildtype CFTR

Cl-

Mutation in CFTR

Healthy neuron Damaged neuron

Sin

gle

Gene

Many G

enes

Rods & cones in healthy retina Rods & cones in RP

Complexity of symptoms makes it difficult to detect rare diseases

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Complex

genetics

Complex

phenotype

Average physician visits before receiving a diagnosis = 7

Average time from symptom onset to accurate diagnosis = 4.8 yrs

Percent rare disease cases that are undiagnosed = ?

Rare diseases are progressive

Faster diagnosis Early intervention Improved quality of life

Source: Engel et al., Journal of Rare Disorders

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Exome sequencing has enhanced our understanding of rare diseases

0

40

80

120

160

2009 2010 2011 2012 2013 2014 2015 2016 2017

• 3,710 genes with

phenotype

causing mutations

in OMIM

• 197,952 mutations

in HGMD

Number of Novel Rare Disease Genes Identified

~130 genes

Source: Boycott et al., Nature Reviews Genetics

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Present since the inception of exome sequencing

Agilent launched the first whole exome sequencing kit

2013 2014 2015 2016 2017 2009 2010 2011 2012

First Whole Exome

Sequencing kit

launched

Exome

customization

enabled

Clinical Research

Exome v2

SureSelect Human

All Exon V6

Focused Exome

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Agilent pioneered whole exome sequencing workflow

DNA

Extraction Library Prep

Target

Enrichment Sequencing

Data

Analysis

SureSelect baits are generated using a high-fidelity oligo synthesis process

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3) Deblock

1) Coupling

2) Oxidation

Repeat n times

Depurination

side reaction

N1 O

O

P O

RO

O

N2 O

O

P O

RO

O

Ni O

O

P O

RO

O

HO

Inkjet

Flood

Long length synthesis is achieved by improved cycle

yield

•↑ Coupling efficiency

•↓ Depurination

•↑ Consistency

RNA

Baits

High fidelity process ensures superior quality baits for target enrichment

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%𝑭𝑳 = (𝑪𝒀 ∗ 𝑫𝒀)𝒏𝒕 %FL= %Full Length CY=Synthesis Cycle Yield DY=Depurination Cycle Yield

Errors per kb Oligo Synthesis Fidelity

No need to QC individual

oligoes

More accurate capture

Agilent SureSelect provides the most versatile platform for target enrichment

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Exomes

Custom panels

Catalog panels

Three pillars that guide Agilent exomes

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Performance Content Flexibility

Agilent SureSelect Exomes

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• Comprehensive exome optimized for rare & inherited disorders

SureSelect Clinical

Research Exome V2 (CREv2)

• Comprehensive exome for translational and clinical research

SureSelect All Human Exon V6

• Targeted exome with optimized coverage of only the disease associated genes

SureSelect Focused Exome

New!

Performance, Content, Flexibity in CREv2

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Performance Content Flexibility

Enhanced coverage

of disease associated

genes

Optimized content

including non-coding

regions associated with

disease

Design customizability

to further enhance your

exome

CREv2 provides enhanced coverage of disease-associated genes

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100x average sequencing depth; 67.3Mb design; 6.5Gb sequencing

5,109 disease-

associated genes

Performance

CREv2 provides high SNP and Indel concordance in targeted regions

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Samples SNP Concordance Indel Concordance

Hom Het

Sample 1 99.91% 99.41% 97.15%

Sample 2 99.91% 99.29% 96.63%

Sample 3 99.91% 99.36% 96.95%

Sample 4 99.93% 99.40% 97.48%

Sample 5 99.95% 99.48% 97.19%

Sample 6 99.91% 99.29% 96.5%

Sample 7 99.95% 99.46% 97%

Sample 8 99.92% 99.42% 97.32%

SNP concordance calculated using HapMap data

Indel concordance calculated using dbSNP data

Agilent exomes provide uniform coverage regardless of GC content

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0

50

100

150

200

1 2 3 4 5 6 7 8 9 10

as.factor(Bin)

Avg

_C

ov

0

50

100

150

200

1 2 3 4 5 6 7 8 9 10

as.factor(Bin)

Avg

_C

ov

Agilent CREv2 Vendor ID

GC High Low GC High Low

0

50

100

150

Pearson’s r = 0.6 Pearson’s r = 0.27 200

0

50

100

150

200

Norm

aliz

ed c

overa

ge

Norm

aliz

ed c

overa

ge

All exomes sequenced to the same average sequencing depth

Exons were divided into deciles based on GC to calculate normalized coverage

Smaller deviation from the mean

across GC bins in CREv2

CREv2 provides consistent coverage in high and low GC regions

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All exomes sequenced to the same average sequencing depth

Agilent CREv2

Vendor ID

41% GC 25% GC

29% GC 45% GC

Agilent CREv2

Vendor ID

Low coverage of

AT-rich exon in

Vendor ID exome

Low coverage of

AT-rich exon in

Vendor ID exome

CREv2 provides the most comprehensive coverage of disease-associated regions

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Disease association information

available with exome!

Optimized coverage of disease-associated genes

Plus

Coverage of splice sites & deep intronic regions

Coverage of other non-coding regions

…….associated with disease

Curated in collaboration with Dr. Madhuri Hegde,

Emory University

Content

CREv2 provides superior disease relevant content

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Agilent CREv2

Vendor ID

Pathogenic variant in 5’ UTR of GJC2

Pathogenic variant associated with leukodystrophy only detectable by Agilent CREv2

but not competitor ID exome

A>G SNV

Mutations that cause retinitis pigmentosa frequently occur in non-coding regions

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Agilent CREv2

Vendor ID

Vendor R

Agilent CREv2

Vendor ID

Vendor R

CREv2 provides more disease-associated regions

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ClinVar

Pathogenic/Likely

Pathogenic

Leukodystrophy

Variants covered

ClinVar

Pathogenic/Likely

Pathogenic Retinitis

Pigmentosa

Variants covered

Agilent CREv2 98.1% 95.3%

Competitor ID 90% 87.9%

Competitor R 90.7% 94.6%

Accelerate the detection of disease-causing mutations with CREv2

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0

10

20

30

40

50

Minimum Average Maximum

Number of Methods

0

5000

10000

15000

20000

Minimum Average Maximum

Cost ($)

Average time to detect leukodystrophy = 8 yrs

Comprehensive coverage of disease associated regions means

Fewer method iterations

Lower cost

Faster detection

Source: Richards et al., Neurology

Build your perfect exome with SureSelect customization capability

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Flexibility

Unmatched flexibility in customization of content and

formats.

Use existing designs as a base to optimize the

exome for your research needs

Copy number changes in rare and inherited disorders

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CNVs account for 10-15% pathogenicity in rare diseases

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10-15% of pathogenic

variants associated with

rare disease are copy

number changes

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

Copy number variants in HGMD Database

Some samples have multiple underlying pathogenic variants

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~5% of samples have multiple pathogenic variants

~12% samples with dual variatns include combination of CNVs and

SNVs

Missed if exome sequencing or CNV analysis performed alone

Detect CNVs, LOH, SNVs &

indels in one NGS assay


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OneSeq Target Enrichment: One Assay, All Variants

1) Evenly spaced genome-wide baits

ClinGen disease

associated regions

ClinGen disease

associated regions

2) High density baits in ClinGen disease associated regions

Gene A Gene B

3) User-defined baits in exonic regions

Copy number

& LOH

SNVs & indels


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OneSeq: Tailored for your needs

OneSeq High Resolution OneSeq Low Resolution

CNV resolution genome-wide 300 kb 1 Mb

CNV resolution in ClinGen

regions 25-50 kb 1 MB

LOH 5 Mb 10 Mb

SNV & Indels

Combine OneSeq CNV

backbone with any

SureSelect exome, ClearSeq

gene panel or SureSelect

custom region

Combine OneSeq CNV

backbone with any

SureSelect exome, ClearSeq

gene panel or SureSelect

custom region

Sequencer recommendation High or medium throughput

sequencers

High, medium or benchtop

sequencers

Region targeted by CNV

backbone 12 Mb 2.7 Mb


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Can OneSeq detect all the important CNVs?

OneSeq 300kb (25-50 kb resolution in ClinGen regions) and 1Mb backbones have

sufficient resolution to detect most CNVs in ClinGen database

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CNVs in ClinGen Database

1) 4,579 CNVs

2) 93% are > 300kb

3) 81% are > 1Mb

Pathogenic

Likely

Pathogenic

Likely

Pathogenic

Likely

Benign

Benign

OneSeq can reliably detect CNVs identified by microarrays

Chromosome Aberration

type

CGH

aberration

size [kb]

OneSeq

aberration size

[kb]

OneSeq avg

log2 ratio

chr13 del 12427 13335 -0.89

chr15 del 2240 1667 -0.37

chr16 del 772 863 -0.43

chr14 amp 987 544 0.54

chr6 amp 370 372 0.61

chr2 del 828 307 -0.46

chr17 amp 163 201 0.49

chr22 amp 172 191 3.00

Detection of 8 CNVs >150 kb with both OneSeq 300 kb and CGH+SNP 4x180K

microarrays in Coriell sample NA08254


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OneSeq can detect intergenic CNVs Duplication upstream of SOX9

Customer

generated

Array data

Case published in Vetro et al, EJHG (2014), 1-8, 1018-4813/14

Microarray

OneSeq can detect CNVs in non-coding regions

OneSeq

4x180K

Catalog design

For Research Use Only. Not for use in diagnostic

procedures.

OneSeq can detect uniparental disomy

Detection of Uniparental Disomy 15 in Coriell

sample NA20409

Copy number

[Log2Ratio]

LOH data

[B allele freq]

CGH+SNP

microarray

confirmation

data

Known common CNV

OneSeq data:

Data analysis bottleneck in NGS workflow

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FASTQ

BAM

VCF

~20,000 variants

Disease-associated variant

Exome Sequencing

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NGS workflow needs substantial compute infrastructure

Sample

prep Library Prep

Target

Enrichment Sequencing

Data

Analysis

Compute intensive

Disparate tools

Time-consuming

Alissa software platform – from raw data to answer

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Make your work flow with Agilent Alissa Clinical Informatics for NGS

One single platform from raw reads to draft lab reports

Comprehensive QC metrics at your fingertips

A team of experts that go the road with you

Agilent NGS solutions for rare and inherited disorders


April 27, 2017

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Sample QC

investigating rare diseases with agilent ngs solutions rare diseases with agilent ngs solutions ......

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