Genome	  in	  a	  Bo*le	  Working	  Group	   Reference	  Material	  (RM)	  Selec:on	  and	  Design	         …	  to	  tell	...
Scope	  of	  Reference	  Material	  Discussion	  •  Human	  Genome	  &	  Tumor	  Sequencing	  •  Variant	  Types	     –  S...
Reference	  Material	  Needed	  For	  •  Clinical	  plaVorm	  valida:on	       –  Sequencing	  System	       –  Bioinforma...
Reference	  Materials	  Are	  Needed	   …	  to	  tell	  the	  truth	  and	  nothing	  but	  …	                            ...
NY	  State	  Guidelines	  –	  Oncology	  NGS	           Minimum	  Data	  Requirement	  -­‐	  Valida:on	  •  Accuracy:	  Se...
Accredita:on	  -­‐	  College	  of	  American	  Pathologists	  (CAP)	  	                                NGS	  Requirements	...
Associa:on	  for	  Molecular	  Pathology	      Comments	  to	  FDA	  UHT-­‐Sequencing	  Mee:ng,	  June	  2011	  •  …	  Per...
Main	  Mee:ngs	  –	  Reference	  Materials	  (RMs)	  •  April	  13,	  2012	  (NIST)	      –  Genome	  in	  a	  Bo*le	  con...
Workgroup	  A*endees	  •  Approximately	  25	  a*endees	     –  Federal,	  incl.	  FDA,	  CDA,	  NIST	     –  Lab	  accred...
Discussion	  Topics	  For	  Human	  Genome	  Sequencing:	  •  What	  sources	  of	  RMs	  to	  consider	        –  Primary...
Reference	  Material	  –	  Intended	  Uses	  •  Characterize	  PlaVorms	  &	  Methods	  	      –  DNA	  sequencing	      –...
Desired	  RM	  Sample	  Characteris:cs	  •  General	  Considera:ons	     –  Sample	  characteris:cs	  are	  more	  importa...
Desired	  RM	  Sample	  Characteris:cs	  (cont.)	  •  High	  Priority	        –  Mul:ple	  ethnici:es	               •  Di...
Desired	  RM	  Sample	  Characteris:cs	  (cont.)	  •  Nice	  to	  have	       –  Interracial	  marriage	  samples	  	     ...
Other	  RM	  Considera:ons	  •  DNA	  from	  low	  passage	  cell	  lines	       –  Understand	  propaga:on	  of	  variant...
RM	  Sample	  Source	  Sugges:ons	  Most	  support	  •  NA12878	         –  Large	  HapMap	  family,	  well	  characterize...
RM	  Sample	  Source	  Sugges:ons	  (cont.)	  Some	  support	  (if	  consent	  sufficient)	  •  HS1011	        –  Charcot	  ...
HapMap	  NA12878	                     An	  Obvious	  Choice?	  •  Mul:tude	  of	  public	  and	  proprietary	  datasets	  ...
HapMap	  NA12878	                                           Consent	  •  Consent	  available	  for	  	      –  Research	  ...
HapMap	  NA12878	                                Status	  as	  RM	  •  NIST	  expects	  first	  batch	  of	  DNA	  from	  C...
Personal	  Genome	  Project	  (PGP)	  Samples	  •  Consent	      –  Research	  and	  commercial	  use	      –  Possibility...
RM:	  Selected	  3	  PGP	  Trios	  Available	  at	  Coriell	  	  •  Ashkenazim	  Jewish	  trio,	  East	  European	  ancest...
PGP	  Info	  -­‐	  hu8E87A9	  (abbreviated)	  https://my.personalgenomes.org/profile/hu8E87A9        | 23
Coriell	  Info	  -­‐	  hu8E87A9	  (abbreviated)	                                                                          ...
Summary	  •  Defined	  required	  RM	  characteris:cs	  •  Ini:al	  set	  of	  RM	  samples	  selected	      –  NA12878	   ...
Contact	  Informa:on	  	  Genome	  in	  a	  Bo*le:	  	  	  h*p://genomeinabo*le.org	  	  Jus:n	  Zook:	  jus:n.zook@nist.g...
Addi:onal	  Informa:on	                                | 27
HapMap	  Re-­‐Consent	  What will happen if I don’t agree to let my sample be used?You will not lose any benefits if you c...
HapMap	  Re-­‐Consent	  The Repository does not let anyone sell material from samples or cell lines.However, information f...
HapMap	  Re-­‐Consent	  … The database will not include any medical information about anyone whose sample isused. It also ...
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Mar2013 Reference Material Selection Working Group

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Mar2013 Reference Material Selection Working Group

  1. 1. Genome  in  a  Bo*le  Working  Group   Reference  Material  (RM)  Selec:on  and  Design   …  to  tell  the  truth  and  nothing  but  …   XGEN  Congress   March  21,  2013   Andrew  Grupe,  PhD  
  2. 2. Scope  of  Reference  Material  Discussion  •  Human  Genome  &  Tumor  Sequencing  •  Variant  Types   –  SNP   –  InDel  /  Subs:tu:on   –  CNV   –  Structural  variant   | 2
  3. 3. Reference  Material  Needed  For  •  Clinical  plaVorm  valida:on   –  Sequencing  System   –  Bioinforma:cs/Analysis  Pipeline  •  Clinical  test  development  and  valida:on   –  Whole  genome   –  Targeted   –  Germline  vs.  tumor  •  Research   –  Process  development  and  QC  •  Product  development   –  Sequencing  Systems   –  SoYware  development   | 3
  4. 4. Reference  Materials  Are  Needed   …  to  tell  the  truth  and  nothing  but  …   | 4
  5. 5. NY  State  Guidelines  –  Oncology  NGS   Minimum  Data  Requirement  -­‐  Valida:on  •  Accuracy:  Sequence  a  well-­‐characterized  reference  sample  (e.g.   HapMap  DNA  GM12878)  to  determine  error  rate  across  all   amplicons.  •  AnalyFcal  sensiFvity:  Establish  the  analy:cal  sensi:vity  of  the   assay  by  interroga:ng  all  variants  in  the  3  amplicons  with  the   consistently  poorest  coverage,  and  all  variants  in  3  amplicons  with   consistently  good  coverage.  This  can  iniFally  be  established  with   defined  mixtures  of  cell  line  DNAs  (not  plasmids),  but  needs  to  be   verified  with  3-­‐5  pa:ent  samples.  •  AnalyFcal  specificity:  Establish  the  analy:cal  specificity  of  the  assay   by  interroga:ng  all  variants  in  the  3  amplicons  with  the  consistently   poorest  coverage,  and  all  variants  in  3  amplicons  with  consistently   good  coverage.  This  can  iniFally  be  established  with  defined   mixtures  of  cell  line  DNAs  (not  plasmids),  but  needs  to  be  verified   with  3-­‐5  pa:ent  samples.   | 5
  6. 6. Accredita:on  -­‐  College  of  American  Pathologists  (CAP)     NGS  Requirements  •  Valida:ons  must  include  informa:on  on  the  analy:cal   target  (examples,  exons,  genes,  exomes,  genomes,  and   transcriptomes).  The  ability  of  the  analy:cal  process  to   sequence  the  target  (e.g.  percentage  of  target   adequately  sequenced)  must  be  described.  •  Valida:ons  must  determine  and  document  analy:cal   sensi:vity,  specificity,  reproducibility,  repeatability  and   precision  for  the  types  of  variants  assayed  (e.g.  single   nucleo:de  variants,  inser:ons  and  dele:ons,   homopolymer  or  repe::ve  sequences).   | 6
  7. 7. Associa:on  for  Molecular  Pathology   Comments  to  FDA  UHT-­‐Sequencing  Mee:ng,  June  2011  •  …  Performance  of  and  coverage  needs  for  a  given  plaVorm   are  likely  to  differ  depending  on  the  nucleic  acid  and  DNA   regions  analyzed,  the  variants  interrogated,  the  rela:ve   allele  propor:ons  of  par:cular  variants,  …  Evalua:on   should  consider  the  effects  of  rela:ve  GC  content,   homopolymeric  and  other  regions  of  repe::ve  sequence,   homologous  gene  regions  and  DNA  structural  variants,  …   This  necessitates  flexibility  and  individualiza:on  in  the   development  of  valida:on  protocols,  guidelines,  and   controls  on  a  (clinical)  applica:on-­‐by-­‐applica:on  basis.  …    •  Assay  controls  should  include  a  range  of  variants,  …   Process  controls  like  NA12876  [sic]  …  and  the  synthe:c   ERCC  RNA  transcripts  from  NIST  are  examples  of  potenFal   standard  reference  materials.  …   | 7
  8. 8. Main  Mee:ngs  –  Reference  Materials  (RMs)  •  April  13,  2012  (NIST)   –  Genome  in  a  Bo*le  consor:um  ini:a:on  •  August  16,  2012  (NIST)   –  Intended  uses  of  RMs   –  RM  selec:on  strategies  •  November  7,  2012  (ASHG)   –  Status  updates  •  December  6,  2012     –  Selec:on  of  ini:al  RMs  •  March  21,  2013  (XGEN  Congress)   | 8
  9. 9. Workgroup  A*endees  •  Approximately  25  a*endees   –  Federal,  incl.  FDA,  CDA,  NIST   –  Lab  accredita:on   –  Clinical  reference  labs   –  PlaVorm  technologies   –  Reference  material  /  reagent  providers   –  Research   | 9
  10. 10. Discussion  Topics  For  Human  Genome  Sequencing:  •  What  sources  of  RMs  to  consider   –  Primary  sample  /  cell  line  •  Consent   –  Available  for  research  and  for  profit  use  •  What  extent  of  prior  characteriza:on  •  Which  ethnici:es,  genders  •  Which  muta:ons  need  to  be  present   –  Is  medical  relevance  necessary  •  Ini:ally  to  have   –  ONE  characterized  genome  RM      -­‐  or   –  Mul:ple  genomes,  lower  level  of  characteriza:on  •  Source  of  commercial  development  and  distribu:on   –  Manufactured  under  quality  system  for  diagnos:c  applica:ons   | 10
  11. 11. Reference  Material  –  Intended  Uses  •  Characterize  PlaVorms  &  Methods     –  DNA  sequencing   –  Exis:ng  &  upcoming  NGS  technologies   –  Research  applica:ons   –  Clinical  diagnos:cs  applica:ons  •  Not  intended  as  reference  material  for   –  Valida:on  of  specific  muta:ons  in  a  panel   | 11
  12. 12. Desired  RM  Sample  Characteris:cs  •  General  Considera:ons   –  Sample  characteris:cs  are  more  important  than   selec:on  of  specific  sample  IDs   –  More  reference  samples  preferred  over  fewer   samples   •  E.g.  prefer  8  fully  characterized  samples  at  high  depth   and  corresponding  trios  at  lower  depth  over  4  fully   characterized  samples  plus  trios   | 12
  13. 13. Desired  RM  Sample  Characteris:cs  (cont.)  •  High  Priority   –  Mul:ple  ethnici:es   •  Diversity  in  structural  varia:on  to  stress  systems   •  However,  no  requirement  for  representa:ves  from  every  ethnic  group   –  Balanced  female  to  male  ra:o   –  Cell  lines,  low  passage   •  Replenish  supply     Targeted  Ethnic  Distribu:on   2  European-­‐ancestry:  northern/western  &  southern/eastern   2  African-­‐American:  AA  &  African,  or  two  AA  from  different  parts  of  the  US   2  La:no:  different  ancestral  places,  US  or  South/Central  America   1  East  Asian   1  South  Asian   | 13
  14. 14. Desired  RM  Sample  Characteris:cs  (cont.)  •  Nice  to  have   –  Interracial  marriage  samples     •  Controlled  admixture   •  Haplotypes  •  Less  cri:cal   –  Phenotypic  characteriza:on   •  Reference  material  not  for  discovery   –  Access  to  RNA  or  :ssues   •  No  limitless  supply  of  material  with  iden:cal  characteris:cs   | 14
  15. 15. Other  RM  Considera:ons  •  DNA  from  low  passage  cell  lines   –  Understand  propaga:on  of  variants  through  cell  line  passaging  •  Modify  DNA  purifica:on  in  future  to  keep  step  with  new  NGS   technologies   –  Current  purified  DNA  fragment  sizes  are  80-­‐100kb   •  OK  for  exis:ng  technologies   –  New  nanopore  technologies  may  need  Mbp  fragments   •  Agarose  embedding  is  proven  extrac:on  technology  •  Consider  footprint  analysis  of  all  batches  prior  to  distribu:on   –  Iden:fy  gene:c  driY,  mix  ups,  ….  ,  develop  benchmarks  •  Reference  material  that  mimics  tumor  sample  characteris:cs   –  FFPE  embedded  cells?  •  Blood  or  saliva  as  primary  (not  cell  line)  DNA  sources   | 15
  16. 16. RM  Sample  Source  Sugges:ons  Most  support  •  NA12878   –  Large  HapMap  family,  well  characterized   –  NIST  contracted  Coriell  for  DNA  batch  •  Personal  Genome  Project  Samples     –  Includes  trios     –  Use  sequence  data  to  derive  admixture   –  h*p://www.personalgenomes.org   –  Consent  includes  research  use,  commercial  use  and  re-­‐iden:fica:on       | 16
  17. 17. RM  Sample  Source  Sugges:ons  (cont.)  Some  support  (if  consent  sufficient)  •  HS1011   –  Charcot  Marie  Tooth  cell  line   •  Lupski  et  al,  NEJM  2010  •  MCF10A     –  Normal  breast   •  Used  by  Horizon  Dx  to  produce  isogenic  cell  lines  with  cancer  relevant  muta:ons  Other  •  African  American  sample  with  70%  sanger  sequence   –  No  cell  line  available   –  Subject  s:ll  alive  =>  re-­‐consent  &  generate  cell  line?  •  huRef  sample   | 17
  18. 18. HapMap  NA12878   An  Obvious  Choice?  •  Mul:tude  of  public  and  proprietary  datasets  •  Cell  line  and  DNA     available  from  Coriell  •  Listed  in  guidelines  as     poten:al  reference     sample  for  clinical  tests   | 18
  19. 19. HapMap  NA12878   Consent  •  Consent  available  for     –  Research  use  HOWEVER  ….  •  Consent  does  not  include   –  Some  commercial  uses   •  Incl.  altera:ons,  re-­‐distribu:on   –  Re-­‐iden:fica:on  through  sequence  data  •  Op:on  to  withdraw  data  and  materials  http://hapmap.ncbi.nlm.nih.gov/downloads/elsi/CEPH_Reconsent_Form.pdfhttp://genomeinabottle.org/forum-topic/what-appropriate-informed-consent-reference-materials-genome-bottle-consortium | 19
  20. 20. HapMap  NA12878   Status  as  RM  •  NIST  expects  first  batch  of  DNA  from  Coriell  in   mid  April  •  Legal  and  IRB  review  at  NIST  for  NA12878  release    •  Start  to  develop  bioinforma:cs  methods  based   on  NA12878  data   –  Have  bioinforma:cs  tools  when  other  samples  are   available   8,000 aliquots of 10ug each on order by NIST from Coriell | 20
  21. 21. Personal  Genome  Project  (PGP)  Samples  •  Consent   –  Research  and  commercial  use   –  Possibility  of  re-­‐iden:fica:on,  including  through  sequence   –  Op:on  to  withdraw  at  any  point   •  Data  removal  and  destruc:on  of  material   www.personalgenomes.org/consent/PGP_Consent_Approved_02212012.pdf    •  Sample  availability   –  Ongoing  enrollment     –  Limited  collec:on  of  ethnically  diverse  trios   h*p://blog.personalgenomes.org/2012/11/29/seeking-­‐diversity/   | 21
  22. 22. RM:  Selected  3  PGP  Trios  Available  at  Coriell    •  Ashkenazim  Jewish  trio,  East  European  ancestry     –  Parents,  Son   –  huAA53EO  /  hu8E87A9  /  hu6E4515  Not  yet  available  at  Coriell    •  East  Asian  trio   –  Parents,  Son   –  hu91BD69  /  hu38168C  /  huCA017E  •  Caucasian  quartet   –  Parents,  2  monozygo:c  twin  daughters   –  huCDC3B8  /  huFE01E1  /  hu1E8957  /  hu961968   | 22
  23. 23. PGP  Info  -­‐  hu8E87A9  (abbreviated)  https://my.personalgenomes.org/profile/hu8E87A9 | 23
  24. 24. Coriell  Info  -­‐  hu8E87A9  (abbreviated)   | 24http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=hu8E87A9
  25. 25. Summary  •  Defined  required  RM  characteris:cs  •  Ini:al  set  of  RM  samples  selected   –  NA12878   •  Many  exis:ng  public  and  proprietary  datasets   •  Listed  in  clinical  guidelines  to  establish  valida:on  parameters   •  Consent  limita:ons   –  Commercial  use,  re-­‐iden:fica:on  through  sequence   •   Under  legal  and  IRB  review  by  NIST   –  Three  PGP  trios   •  One  trio  already  available  at  Coriell  •  Consent  without  withdrawal  op:on  may  not  meet   ethical  review  standards   | 25
  26. 26. Contact  Informa:on    Genome  in  a  Bo*le:      h*p://genomeinabo*le.org    Jus:n  Zook:  jus:n.zook@nist.gov  Marc  Salit:  salit@nist.gov      Andrew  Grupe:      andrew.grupe@celera.com   | 26
  27. 27. Addi:onal  Informa:on   | 27
  28. 28. HapMap  Re-­‐Consent  What will happen if I don’t agree to let my sample be used?You will not lose any benefits if you choose not to let your sample be used. Ifyou don’t agree to let your sample be used, it will not be used for the HapMap.However, it will continue to be used for other IRB approved research studies,just as it has been in the past, unless you specifically tell us that you don’t wantit used for such studies anymore.Can I change my mind after I agree to let my sample be used?Deciding whether to let your sample be used for the HapMap is completely upto you. You will not lose any benefits if you choose not to let your sample beused. However, once your sample has been studied and your geneticinformation has been put in the database, you will not be able to take thatinformation back. | 28
  29. 29. HapMap  Re-­‐Consent  The Repository does not let anyone sell material from samples or cell lines.However, information from genetics research sometimes helps companiesmake products to diagnose or treat diseases. If information from your family’scell lines leads to making a product, it would probably contribute only in a verysmall way. Also, because the cell lines will not have names on them, neither theresearchers nor anyone at the Repository would know if your samples wereeven used. So you will not get any additional payment for having your sampleused in this project. | 29
  30. 30. HapMap  Re-­‐Consent  … The database will not include any medical information about anyone whose sample isused. It also will not include any information that could identify who the individual peopleor families are. …Because the database will be public, people who do identity testing, such as for paternitytesting or law enforcement, may also use the samples, the database, and the HapMap, todo general research. However, it will be very hard for anyone to learn anything about youpersonally from any of this research because none of the samples, the database, or theHapMap will include your name or any other information that could identify you or yourfamily.What are the risks of having my sample used for this project?If your family’s samples are used, lots of genetic information from your samples will be putin the database, and lots of people will be able to look at it for any purpose. However,there are only a couple of ways anybody could trace the information back to you. One is ifthey thought your information might bein the database, got another sample from you, did many tests on that sample, and thencompared the genetic information from those tests with the information in the database.The other is if somebody compared the information in the database with geneticinformation known to be from you that was in another database and figured out who youwere. The risk of either of these things happening is very small, but it may grow in thefuture.We cannot always predict the results of research, so new risks to you may come up in thefuture that we can’t predict now. | 30

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