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  • 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

    Research use only. Not for use in diagnostic purposes.

    2

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    3

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    4

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    5

    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

  • April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    6

    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

  • April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    7

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    8

  • April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    9

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    10

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    11

    % = ( ) %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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    12

    Exomes

    Custom panels

    Catalog panels

  • Three pillars that guide Agilent exomes

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    13

    Performance Content Flexibility

  • Agilent SureSelect Exomes

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    14

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    15

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    16

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    17

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    18

    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

    Pearsons r = 0.6 Pearsons 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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    19

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    20

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    21

    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

    April 27, 2017

    Research use only. Not for use in diagnostic purposes.

    22

    Agilent CREv2

    Vendor ID

    Vendor R

    Agilent CREv2

    Vendor ID

    Vendor R

  • CREv2 provides more disease-associate

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