© Personalis, Inc. All Rights Reserved.
Dissecting the missing
diagnostic yield in exome
sequening
Deanna M. Church, Perso...
© Personalis, Inc. All Rights Reserved. 2
Disclosure
I work for Personalis, Inc. A company that provides whole
genome and ...
© Personalis, Inc. All Rights Reserved. 3
Acknowledgements
Personalis
Jason Harris
Sarah Garcia
Jeane Tirsch
Mark Pratt
Sc...
© Personalis, Inc. All Rights Reserved. 4
Diagnostic Yield
Clinically informative results
returned to clinician
Total numb...
© Personalis, Inc. All Rights Reserved. 5
Diagnostic Yield
ACMG Recommendations for interpreting and reporting sequencing ...
© Personalis, Inc. All Rights Reserved. 6
Diagnostic Yield
Pathogenic
Likely Pathogenic
Unknown Significance
Likely Benign...
© Personalis, Inc. All Rights Reserved. 7
Diagnostic Yield: Karyotyping
1956: Total number of chromosomes (Tijo and Levan)...
© Personalis, Inc. All Rights Reserved. 8
Diagnostic Yield: FISH
1981: FISH (Langer et al.)
8-11%
© Personalis, Inc. All Rights Reserved. 9
Diagnostic Yield: CGH
1998: BAC aCGH (Pinkel et al.)
2003-2004: oligo aCGH (many...
© Personalis, Inc. All Rights Reserved. 10
Diagnostic Yield
Adapted from http://www.ndsu.edu/pubweb/~mcclean/plsc431/
home...
© Personalis, Inc. All Rights Reserved. 11
Diagnostic Yield: Gene Panels
http://www.ncbi.nlm.nih.gov/gtr/
Sanger sequencin...
© Personalis, Inc. All Rights Reserved. 12
Diagnostic Yield: Gene Panels
10% 100%
GLA
Non-Syndomic
Hearing Loss
Long-QT
No...
© Personalis, Inc. All Rights Reserved. 13
Diagnostic Yield: Exome
25-56%
Solved
56%
Unsolved
44%
Diagnostic Yield
Yang et...
© Personalis, Inc. All Rights Reserved. 14
Diagnostic Yield: Exome
Genes referenced in ClinVar vs. Genes referenced in GTR
© Personalis, Inc. All Rights Reserved. 15
Diagnostic Yield
Analytical Validity
Clinical Validity
Our ability to reliably ...
© Personalis, Inc. All Rights Reserved. 16
Diagnostic Yield
© Personalis, Inc. All Rights Reserved. 17
Missing Diagnostic Yield
Analytical Validity
© Personalis, Inc. All Rights Reserved. 18
Standard Exomes and Genomes are not “Finished”
© Personalis, Inc. All Rights Reserved. 19
Standard Exomes and Genomes are not “Finished”
http://www.bioplanet.com/gcat
© Personalis, Inc. All Rights Reserved. 20
Standard Exomes and Genomes are not “Finished”
http://www.bioplanet.com/gcat
© Personalis, Inc. All Rights Reserved. 21
Standard Exomes Don’t Cover the Whole Exome
ACE Clinical Exome
Standard Exome
M...
© Personalis, Inc. All Rights Reserved. 22
Standard Exomes Don’t Cover the Whole Exome
NM_003002.3 (reference transcript)
...
© Personalis, Inc. All Rights Reserved. 23
© Personalis, Inc. All Rights Reserved. 24
Whole Genome Sequencing Does Not Completely Solve the
Accuracy Challenges
ACE C...
© Personalis, Inc. All Rights Reserved. 25
Standard Genomes Don’t Cover the Whole Genome
PMS2
Lynch syndrome
CDK11A
Neurob...
© Personalis, Inc. All Rights Reserved. 26
Diagnostic Yield
ACMG Recommendations for interpreting and reporting sequencing...
© Personalis, Inc. All Rights Reserved. 27
Most of what we know is in the context of genes
ClinVar: 55,467 variants*
* Wit...
© Personalis, Inc. All Rights Reserved. 28
Standard Exomes and Genomes are not “Finished”
WGS cost > WES cost.
Still need ...
© Personalis, Inc. All Rights Reserved. 29
Reference Genomes Aren’t Finished
GRCh37
© Personalis, Inc. All Rights Reserved. 30
Reference Genomes Aren’t Finished
GRCh38 is coming
(September, 2013)
© Personalis, Inc. All Rights Reserved. 31
Reference Genomes Aren’t Finished
http://www.ncbi.nlm.nih.gov/assembly/GCF_0000...
© Personalis, Inc. All Rights Reserved. 32
Reference Genomes Aren’t Finished
GRCh38
© Personalis, Inc. All Rights Reserved. 33
Reference Genomes Aren’t Finished
GRCh37GRCh38
© Personalis, Inc. All Rights Reserved. 34
Migrating to GRCh38
NCBI Remap tool
http://www.ncbi.nlm.nih.gov/genome/tools/re...
© Personalis, Inc. All Rights Reserved. 35
Migrating to GRCh38
GRCh38
GRCh37
Collapsed Sequence by Assembly
© Personalis, Inc. All Rights Reserved. 36
Migrating to GRCh38
GRCh38
GRCh37
Expanded Sequence by Assembly
© Personalis, Inc. All Rights Reserved. 37
GRCh38 annotation (NCBI)
No dbSNP 141 variants
Migrating to GRCh38
© Personalis, Inc. All Rights Reserved. 38
First Pass remap Second Pass remap
Migrating to GRCh38
© Personalis, Inc. All Rights Reserved. 39
GRCh38
Alternate Loci
Migrating to GRCh38
© Personalis, Inc. All Rights Reserved. 40
Migrating to GRCh38
https://www.eventbrite.com/e/grc-assembly-workshop-at-
geno...
© Personalis, Inc. All Rights Reserved. 41
Migrating to GRCh38
652 genes on FIX patches
in GRCh37.p13
Fix Patch
Novel Patch
© Personalis, Inc. All Rights Reserved. 42
Migrating to GRCh38
GRCh37.p13: NC_000001.10 (chr1)
NCBI Annotation Run 105
GRC...
© Personalis, Inc. All Rights Reserved. 43
Migrating to GRCh38
SHANK2*
* on 20 tests in GTR
Fix Patch
Chromosome
© Personalis, Inc. All Rights Reserved. 44
Migrating to GRCh38: using Fix patches
© Personalis, Inc. All Rights Reserved. 45
Migrating to GRCh38: using Fix patches
hs37d5
Fix patch
© Personalis, Inc. All Rights Reserved. 46
Migrating to GRCh38: using Fix patches
hs37d5
Fix patch
© Personalis, Inc. All Rights Reserved. 47
Migrating to GRCh38: using Fix patches
GRCh37 vs. Fix Patch
GRCh38
© Personalis, Inc. All Rights Reserved. 48
Migrating to GRCh38: using Fix patches
© Personalis, Inc. All Rights Reserved. 49
Migrating to GRCh38: using Fix patches
© Personalis, Inc. All Rights Reserved. 50
Missing Diagnostic Yield
Clinical Validity
© Personalis, Inc. All Rights Reserved. 51
Diagnostic Yield
ACMG Recommendations for interpreting and reporting sequencing...
© Personalis, Inc. All Rights Reserved. 52
Integrating the literature
Adapted from http://www.ndsu.edu/pubweb/~mcclean/pls...
© Personalis, Inc. All Rights Reserved. 53
1994
Integrating the literature
© Personalis, Inc. All Rights Reserved. 54
Piton et al., 2013
XLID-Causing Mutations and Associated
Genes Challenged in th...
© Personalis, Inc. All Rights Reserved. 55
Integrating the literature
© Personalis, Inc. All Rights Reserved. 56
Integrating the literature
© Personalis, Inc. All Rights Reserved. 57
Evaluating Variants: ClinVar
Based on variant_summary.txt
(May 20, 2014)
© Personalis, Inc. All Rights Reserved. 58
Figure 1b. 1000 Genomes Consortium, Nature 2012
Rare variants
© Personalis, Inc. All Rights Reserved. 59
Communicating knowledge
ATM: 5762ins137
“137 nt added between exons 40 and 41”
...
© Personalis, Inc. All Rights Reserved. 60
Exon 38 Exon 39
Communicating knowledge
© Personalis, Inc. All Rights Reserved. 61
C>G
NC_000011.9 (chr11) 108179684
Communicating knowledge
© Personalis, Inc. All Rights Reserved. 62
Turk et al., 2013
c.82C>T; p.R28WLMNA:
Communicating knowledge
© Personalis, Inc. All Rights Reserved. 63
Communicating knowledge
ClinVar Variant Genomic Locations
© Personalis, Inc. All Rights Reserved. 64
NCBI Homo sapiens annotation run 105
Ensembl 74
Communicating knowledge
© Personalis, Inc. All Rights Reserved. 65
Improving Diagnostic Yield
More Sequence
Better Phenotyping
More precise data h...
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Church SFAF2014 keynote

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Keynote lecture from the Sequencing, Finishing and Analysis in the Future meeting, Santa Fe, NM 2014

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  • This is not the specific workflow used by every lab, but rather outlines the formal framework that clinical labs should use when classifying variants. Additionally if labs are CAP or CLIA certified there are reams of accompanying documentation of their process and a testing framework must be in place to show reproducibility. The ACMG continues to refine these recommendations and a new set of guidelines will be released soon. This is in stark contrast to research labs, where there is no single framework used to classify variants.
  • Is the 5 tier still in place- or is it three tier now?
  • Original genomic tests were often specified for a non-specific phenotype (like developmental delay) and the technology of the time did not allow for the identification of small scale events.
  • If a disease doesn’t affect a lot of people, there won’t be a specific test available. It is also difficult to o keep tests up to date as many groups are reporting on variants in genes that have only been associated with disease in the last couple of years.
  • Slide- difference between discovery and research
  • Susceptibility to thyrotoxic periodic paralysis
  • Add number of genes on fix patches (and only on fix patches and alt loci)- maybe show an example- SRGAP!
  • Church SFAF2014 keynote

    1. 1. © Personalis, Inc. All Rights Reserved. Dissecting the missing diagnostic yield in exome sequening Deanna M. Church, Personalis May 29, 2014 @deannachurch
    2. 2. © Personalis, Inc. All Rights Reserved. 2 Disclosure I work for Personalis, Inc. A company that provides whole genome and whole exome sequencing and analysis services.
    3. 3. © Personalis, Inc. All Rights Reserved. 3 Acknowledgements Personalis Jason Harris Sarah Garcia Jeane Tirsch Mark Pratt Scott Kirk Rich Chen John West Genome Reference Consortium NCBI Valerie Schneider Terence Murphy Donna Maglott Melissa Landrum Wendy Rubinstein Jennifer Lee
    4. 4. © Personalis, Inc. All Rights Reserved. 4 Diagnostic Yield Clinically informative results returned to clinician Total number of tests performed
    5. 5. © Personalis, Inc. All Rights Reserved. 5 Diagnostic Yield ACMG Recommendations for interpreting and reporting sequencing variants
    6. 6. © Personalis, Inc. All Rights Reserved. 6 Diagnostic Yield Pathogenic Likely Pathogenic Unknown Significance Likely Benign Benign
    7. 7. © Personalis, Inc. All Rights Reserved. 7 Diagnostic Yield: Karyotyping 1956: Total number of chromosomes (Tijo and Levan) 1959: Trisomy 21 (Lejeune) 3-5%
    8. 8. © Personalis, Inc. All Rights Reserved. 8 Diagnostic Yield: FISH 1981: FISH (Langer et al.) 8-11%
    9. 9. © Personalis, Inc. All Rights Reserved. 9 Diagnostic Yield: CGH 1998: BAC aCGH (Pinkel et al.) 2003-2004: oligo aCGH (many) 11-18%
    10. 10. © Personalis, Inc. All Rights Reserved. 10 Diagnostic Yield Adapted from http://www.ndsu.edu/pubweb/~mcclean/plsc431/ homework/positional-cloning/ 1990 2000 2010 APC Aniridia Fragile X Myotonic Dystrophy Norrie Disease Huntington Disease Menkes Disease NF2 Tuberous Sclerosis Achondroplasia BRCA1 PKD1 Ataxia Telangiectasia Bloom syndrome SMA Hemochromatosis Long QT PKD2 Treacher Collins Alagille Syndrome Angelman Deafness 1 Wolfram Retinis Pigmentosa Deafness 5
    11. 11. © Personalis, Inc. All Rights Reserved. 11 Diagnostic Yield: Gene Panels http://www.ncbi.nlm.nih.gov/gtr/ Sanger sequencing of entire coding sequence: 9,589 NGS sequencing of entire coding sequence: 1,451
    12. 12. © Personalis, Inc. All Rights Reserved. 12 Diagnostic Yield: Gene Panels 10% 100% GLA Non-Syndomic Hearing Loss Long-QT Noonan Alagille (JAG1) Laboratory for Molecular Medicine Lieve et al., 2013
    13. 13. © Personalis, Inc. All Rights Reserved. 13 Diagnostic Yield: Exome 25-56% Solved 56% Unsolved 44% Diagnostic Yield Yang et al., 2013 Numerous ACMG abstracts Personalis Data (56 cases) Numbers reported across labs
    14. 14. © Personalis, Inc. All Rights Reserved. 14 Diagnostic Yield: Exome Genes referenced in ClinVar vs. Genes referenced in GTR
    15. 15. © Personalis, Inc. All Rights Reserved. 15 Diagnostic Yield Analytical Validity Clinical Validity Our ability to reliably identify variants Our ability to reliably associate variants with a disease.
    16. 16. © Personalis, Inc. All Rights Reserved. 16 Diagnostic Yield
    17. 17. © Personalis, Inc. All Rights Reserved. 17 Missing Diagnostic Yield Analytical Validity
    18. 18. © Personalis, Inc. All Rights Reserved. 18 Standard Exomes and Genomes are not “Finished”
    19. 19. © Personalis, Inc. All Rights Reserved. 19 Standard Exomes and Genomes are not “Finished” http://www.bioplanet.com/gcat
    20. 20. © Personalis, Inc. All Rights Reserved. 20 Standard Exomes and Genomes are not “Finished” http://www.bioplanet.com/gcat
    21. 21. © Personalis, Inc. All Rights Reserved. 21 Standard Exomes Don’t Cover the Whole Exome ACE Clinical Exome Standard Exome Mutations in RPGR cause ~80% of X-linked retinitis pigmentosa Read depth Sensitivity to detect heterozygotes
    22. 22. © Personalis, Inc. All Rights Reserved. 22 Standard Exomes Don’t Cover the Whole Exome NM_003002.3 (reference transcript) NM_001276506.1 (new RefSeq Transcript)
    23. 23. © Personalis, Inc. All Rights Reserved. 23
    24. 24. © Personalis, Inc. All Rights Reserved. 24 Whole Genome Sequencing Does Not Completely Solve the Accuracy Challenges ACE Clinical Exome™ (12G) Standard Whole Genome (100G) - Dark orange represents coverage at 1 sigma from mean >25x coverage (required to call heterozygous SNVs and indels accurately) Previously described variants Depth Coverage Plot of RPGR
    25. 25. © Personalis, Inc. All Rights Reserved. 25 Standard Genomes Don’t Cover the Whole Genome PMS2 Lynch syndrome CDK11A Neuroblastoma FLG Ichthyosis Vulgaris (Nat Gen 2007)
    26. 26. © Personalis, Inc. All Rights Reserved. 26 Diagnostic Yield ACMG Recommendations for interpreting and reporting sequencing variants
    27. 27. © Personalis, Inc. All Rights Reserved. 27 Most of what we know is in the context of genes ClinVar: 55,467 variants* * With a genomic location as of 03/07/2014 High quality Exons Any CDS Variants with no overlap Distance of non-overlapping variants to closest high quality exon feature
    28. 28. © Personalis, Inc. All Rights Reserved. 28 Standard Exomes and Genomes are not “Finished” WGS cost > WES cost. Still need to supplement WGS to get full gene coverage. Most interpretation is in the context of genes.
    29. 29. © Personalis, Inc. All Rights Reserved. 29 Reference Genomes Aren’t Finished GRCh37
    30. 30. © Personalis, Inc. All Rights Reserved. 30 Reference Genomes Aren’t Finished GRCh38 is coming (September, 2013)
    31. 31. © Personalis, Inc. All Rights Reserved. 31 Reference Genomes Aren’t Finished http://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/
    32. 32. © Personalis, Inc. All Rights Reserved. 32 Reference Genomes Aren’t Finished GRCh38
    33. 33. © Personalis, Inc. All Rights Reserved. 33 Reference Genomes Aren’t Finished GRCh37GRCh38
    34. 34. © Personalis, Inc. All Rights Reserved. 34 Migrating to GRCh38 NCBI Remap tool http://www.ncbi.nlm.nih.gov/genome/tools/remap First Pass Alignments Second Pass Alignments First Pass + Second Pass = Collapse Second Pass Only = Expansion
    35. 35. © Personalis, Inc. All Rights Reserved. 35 Migrating to GRCh38 GRCh38 GRCh37 Collapsed Sequence by Assembly
    36. 36. © Personalis, Inc. All Rights Reserved. 36 Migrating to GRCh38 GRCh38 GRCh37 Expanded Sequence by Assembly
    37. 37. © Personalis, Inc. All Rights Reserved. 37 GRCh38 annotation (NCBI) No dbSNP 141 variants Migrating to GRCh38
    38. 38. © Personalis, Inc. All Rights Reserved. 38 First Pass remap Second Pass remap Migrating to GRCh38
    39. 39. © Personalis, Inc. All Rights Reserved. 39 GRCh38 Alternate Loci Migrating to GRCh38
    40. 40. © Personalis, Inc. All Rights Reserved. 40 Migrating to GRCh38 https://www.eventbrite.com/e/grc-assembly-workshop-at- genome-informatics-tickets-11194490021
    41. 41. © Personalis, Inc. All Rights Reserved. 41 Migrating to GRCh38 652 genes on FIX patches in GRCh37.p13 Fix Patch Novel Patch
    42. 42. © Personalis, Inc. All Rights Reserved. 42 Migrating to GRCh38 GRCh37.p13: NC_000001.10 (chr1) NCBI Annotation Run 105 GRCh37.p13: NW_003871056.3 (chr1 fix patch)
    43. 43. © Personalis, Inc. All Rights Reserved. 43 Migrating to GRCh38 SHANK2* * on 20 tests in GTR Fix Patch Chromosome
    44. 44. © Personalis, Inc. All Rights Reserved. 44 Migrating to GRCh38: using Fix patches
    45. 45. © Personalis, Inc. All Rights Reserved. 45 Migrating to GRCh38: using Fix patches hs37d5 Fix patch
    46. 46. © Personalis, Inc. All Rights Reserved. 46 Migrating to GRCh38: using Fix patches hs37d5 Fix patch
    47. 47. © Personalis, Inc. All Rights Reserved. 47 Migrating to GRCh38: using Fix patches GRCh37 vs. Fix Patch GRCh38
    48. 48. © Personalis, Inc. All Rights Reserved. 48 Migrating to GRCh38: using Fix patches
    49. 49. © Personalis, Inc. All Rights Reserved. 49 Migrating to GRCh38: using Fix patches
    50. 50. © Personalis, Inc. All Rights Reserved. 50 Missing Diagnostic Yield Clinical Validity
    51. 51. © Personalis, Inc. All Rights Reserved. 51 Diagnostic Yield ACMG Recommendations for interpreting and reporting sequencing variants
    52. 52. © Personalis, Inc. All Rights Reserved. 52 Integrating the literature Adapted from http://www.ndsu.edu/pubweb/~mcclean/plsc431/ homework/positional-cloning/ 1990 2000 2010 APC Aniridia Fragile X Myotonic Dystrophy Norrie Disease Huntington Disease Menkes Disease NF2 Tuberous Sclerosis Achondroplasia BRCA1 PKD1 Ataxia Telangiectasia Bloom syndrome SMA Hemochromatosis Long QT PKD2 Treacher Collins Alagille Syndrome Angelman Deafness 1 Wolfram Retinis Pigmentosa Deafness 5 2012 GO-ESP 1000G Phase 1
    53. 53. © Personalis, Inc. All Rights Reserved. 53 1994 Integrating the literature
    54. 54. © Personalis, Inc. All Rights Reserved. 54 Piton et al., 2013 XLID-Causing Mutations and Associated Genes Challenged in the Light of Data from Large-Scale Exome Sequencing 10/106: questioned 15/106: need replication
    55. 55. © Personalis, Inc. All Rights Reserved. 55 Integrating the literature
    56. 56. © Personalis, Inc. All Rights Reserved. 56 Integrating the literature
    57. 57. © Personalis, Inc. All Rights Reserved. 57 Evaluating Variants: ClinVar Based on variant_summary.txt (May 20, 2014)
    58. 58. © Personalis, Inc. All Rights Reserved. 58 Figure 1b. 1000 Genomes Consortium, Nature 2012 Rare variants
    59. 59. © Personalis, Inc. All Rights Reserved. 59 Communicating knowledge ATM: 5762ins137 “137 nt added between exons 40 and 41” McConville et al., 1996
    60. 60. © Personalis, Inc. All Rights Reserved. 60 Exon 38 Exon 39 Communicating knowledge
    61. 61. © Personalis, Inc. All Rights Reserved. 61 C>G NC_000011.9 (chr11) 108179684 Communicating knowledge
    62. 62. © Personalis, Inc. All Rights Reserved. 62 Turk et al., 2013 c.82C>T; p.R28WLMNA: Communicating knowledge
    63. 63. © Personalis, Inc. All Rights Reserved. 63 Communicating knowledge ClinVar Variant Genomic Locations
    64. 64. © Personalis, Inc. All Rights Reserved. 64 NCBI Homo sapiens annotation run 105 Ensembl 74 Communicating knowledge
    65. 65. © Personalis, Inc. All Rights Reserved. 65 Improving Diagnostic Yield More Sequence Better Phenotyping More precise data handling

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