140128 use cases of giab RMs

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140128 use cases of giab RMs

  1. 1. Use  cases  of  GIAB  Reference   Materials   NIST,  HSPH,  Claritas,  NCI,  MSSM,   Personalis,  Qiagen   Genome  in  a  Bo>le  Consor?um  
  2. 2. Preliminary  uses  of  high-­‐confidence   NIST-­‐GIAB  genotypes  for  NA12878   •  NIST  has  released  several  versions  of  high-­‐ confidence  genotypes  for  its  pilot  RM   •  We’ve  collected  some  examples  of  how   people  are  using  these  genotypes  
  3. 3. •  •  is pharmacogenomics — the use ample, three recently published a step fu of genomic information to iden- clinical trials raise questions consider tify the right drug at the right about the clinical utility of using conditio dose for each patient. More than pharmacogenetic information in not, in 120 FDA-approved drugs have the initial dosing of vitamin K premium pharmacogenomics information anatagonists.3 however, in their labeling, providing imThe FDA based its decision to So  you’ve  sequenced  my   differences grant marketing authorization for of genom portant details about insuranc genome.  How  well  did  the drug and, in the Illumina instrument platform ance, or you   in response to do?   some cases, recommending ge- and reagents on their demonThe l netic testing before prescribing.2 strated accuracy across numer- use of g –  FDA  approval…   NIST  work  omacogenomics potential of phar- ous human chromosomes. Preci- medicine n  But the full is largely unreal- 19 genomic segments, spanning of this developing   the  most  accurate   of the logistic chal- sion and reproducibility across Court ru ized, because interpreta?on  of  a  human   suitable instruments, users, days, and re- lecular Pa lenges in obtaining ENGLA ND were also demonstrated. that isol genomic information in a genome,  coupled  with  The NEWtimely agent lots JOURNAL of MEDICINE a   enough fashion to guide The marketing authorization of DNA ca NIST  Reference  Material   prescribing. Placing genomic information platform for clinicurrently  being  delectronic medical record a sequencingprobably expand the decision eveloped,   in the cal use will access to enabled  FDA  to  assess  the   of per- incorporation of genetic informa- but also would facilitate this kind performance  of  the   sonalized medicine. If the pa- tion into health care. But even for the in sequencer  stient’s entire genome were part the most promising technologies quencing ubmi>ed  for   marke?ng  aof his or her medical record, cannot fully realize their poten- fore the pproval.   NIST  work  with  FDA  helps  answer  the   ques?on…   Perspective Paper  Describing  NIST-­‐GIAB   n JOURNAL of The NEW ENGLA NDengl j med nejm.org MEDICINE 2 Characteriza?on  of  NA12878:   First FDA Authorization for Next-Generation Sequencer Nature  Biotechnology,  accepted   The New England Journal of Medicine Francis S. Collins, M.D., Ph.D., and Margaret A. Hamburg, M.D. Downloaded from DOI:  arXiv:1307.4661  [q-­‐bio.GN]   nejm.org at FDA Biosciences Library on November 20, 2013. For personal use only. No
  4. 4. HSPH  –  Brad  Chapman     Comparing  variant  callers   h>p://bcbio.wordpress.com/2013/10/21/updated-­‐comparison-­‐of-­‐variant-­‐detec?on-­‐ methods-­‐ensemble-­‐freebayes-­‐and-­‐minimal-­‐bam-­‐prepara?on-­‐pipelines/  
  5. 5. Freebayes  SNP  calls  changed  very  li>le  in  2013   h>p://www.bioplanet.com/gcat/reports/1933-­‐westleouzm/variant-­‐calls/illumina-­‐100bp-­‐pe-­‐exome-­‐150x/bwamem-­‐ freebayes-­‐0-­‐9-­‐10-­‐131226/compare-­‐1934-­‐akckizzzfr-­‐1931-­‐laqgzjytqw-­‐1935-­‐xwckffckoa/snp/group-­‐quality  
  6. 6. Freebayes  indel  calls  improved  in  2013   h>p://www.bioplanet.com/gcat/reports/1933-­‐westleouzm/variant-­‐calls/illumina-­‐100bp-­‐pe-­‐exome-­‐150x/bwamem-­‐ freebayes-­‐0-­‐9-­‐10-­‐131226/compare-­‐1934-­‐akckizzzfr-­‐1931-­‐laqgzjytqw-­‐1935-­‐xwckffckoa/indel/group-­‐quality  
  7. 7. Feedback  from  MoCha  lab  in  NCI     •  We  built  a  targeted  amplicons  NGS  assay  for   detec?ng  muta?ons  in  clinical  tumor  specimens   •  To  assess  the  assay’s  specificity,  we  compared  84   runs  of  CEPH  NA12878  data  from  our  assay  with   NIST’s  consensus  variant  list  (VCF  v2.15)     •  We  observed  a  high  overall  concordance  with  a   few  FP  variants  in  homopolymeric  regions  unique   in  our  plahorm   •  We  concluded  that  NIST  GIAB  is  a  useful   reference  standard  to  evaluate  assay  specificity  
  8. 8. Personalis  –  Categorizing  exome  regions   h>p://www.personalis.com/assets/files/posters/ashg2013/Towards_a_medical-­‐grade_exome.pdf  
  9. 9. Genome  in  a  Bo>le  @   Mount  Sinai   Michael  Linderman     Icahn  Ins?tute  for  Genomics  and  Mul?scale  Biology   Dept.  of  Gene?cs  and  Genomic  Sciences  
  10. 10. Ongoing  clinical  pipeline  valida?on     Reference  Materials   SNP  Array   Technical  replicates:   NA12878   NA12891   NA12892   NA18507   NA10080   Sanger   Concordance  Analysis   Genome  in   a  Bo>le   Pla?num   Genomes  
  11. 11. Evalua?ng  and  tuning  variant  calling  &   filtering   Measure  overall  analy?cal   performance   Tune  VQSR  threshold  senng  to  inflec?on  point   We  evaluate  a  set  of  NA12878  technical  replicates  against  GIAB  for  each  new   pipeline  version  
  12. 12. GIAB  Use  at  Qiagen  (Frederick,  MD)   •  Use  GIAB  false  posi?ve  sites  to  quickly  iden?fy   PCR  ar?facts  in  reads  from  PCR-­‐enriched   samples.   •  Compare  accuracy  of  PCR-­‐enrichment   amplicon  sequencing  to  accuracy  of   hybridiza?on-­‐capture  whole-­‐exome   sequencing.   •  Tune  variant  calling  pipeline  for  good  balance   between  sensi?vity  and  specificity.   •  Compare  variant  calling  methods,  of  course!    
  13. 13. iden?fy  PCR  ar?facts  quickly   •  example:  sequenced  fragment  was  primed  by  a  PCR  primer-­‐ dimer  strand  formed  in  earlier  PCR  cycles   5-GGACCTGTGGGTGGGTAAC-3 oligo intended for chr1 locus   |||||||||||xxxx   3-GACACCCACCCGAGGTT-5 oligo intended for chr5 locus   5-GGGTCTGTGGGTGGGCTCCAA-3 DNA sample (chr5 locus) || |||||||||||||||||   3-CCTGGACACCCACCCGAGGTT-5 dimer product primes 4 bp upstream AC false positive variant called  
  14. 14. compare  amplicon  enrichment  to   hybridiza?on  capture  enrichment   •  characterize  panels  for  mul?plex-­‐PCR  enrichment   •  compare  to  exome  capture  read  set  from  Mount  Sinai   Medical  School  (105x  coverage,  36.6Mb)   read  set   indel   TPR   PPV   GeneRead  662  Kb   46.9   0.94   0.92   Mount  Sinai  37  Mb   snp   FPR  x  1E6   7.6   0.89   0.99   GeneRead  662  Kb   9.1   0.60   0.79   Mount  Sinai  37Mb   0.7   0.75   0.98  
  15. 15. tune  variant  calling   pipelines   •  generate  ROC  curves  to   compare  Strelka  and  MuTect   matched  tumor/normal   •  “tumor”  here  is  8,  16,  36,  and   100%  spike-­‐in  of  NA12878  DNA  
  16. 16. NIST GIAB Confident Calls as the Gold Standard ●  Community resource to which all can contribute and access ○  Group-specific or internally-generated gold standards have unknown methods, origin, and curation. ○  NIST GIAB confident calls allows public access and contributions. ●  Claritas Genomics uses this callset for: ●  Technology feasibility, research and development ○  test new technologies, protocols, reagents, methods, etc. ●  Validation and verification ○  assay validation and verification for clinical use ●  Critical attributes: ○  a large number of confident true negative positions! ○  confident region allows for scale or genome-scale sensitivity and specificity analysis. ○  part of a large well-characterized pedigree ○  a trusted source of truth CONFIDENTIAL CLARITAS GENOMICS
  17. 17. Other  use  cases?   •  What  other  types  of  uses  might  people   develop?  

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