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  

Scope  of  Reference  Material  Discussion  

•  Human  Genome  &  Tumor  Sequencing  

•  Variant  Types  – SNP  –  InDel  /  Subs:tu:on  – CNV  – Structural  variant  

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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  

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Reference  Materials  Are  Needed  …  to  tell  the  truth  and  nothing  but  …  

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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.  

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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).  

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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.  …  

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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)  

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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  

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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  

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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  

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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  

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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

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  

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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  

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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  

   

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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  

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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  

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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  

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http://genomeinabottle.org/forum-topic/what-appropriate-informed-consent-reference-materials-genome-bottle-consortium

http://hapmap.ncbi.nlm.nih.gov/downloads/elsi/CEPH_Reconsent_Form.pdf

•  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  

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HapMap  NA12878  Status  as  RM  

8,000 aliquots of 10ug each on order by NIST from Coriell

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/  

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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  

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PGP  Info  -­‐  hu8E87A9  (abbreviated)  

| 23 https://my.personalgenomes.org/profile/hu8E87A9

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Coriell  Info  -­‐  hu8E87A9  (abbreviated)  

http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=hu8E87A9

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  

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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  

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Addi:onal  Informa:on  

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HapMap  Re-­‐Consent  

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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. If you 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 want it 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 up to you. You will not lose any benefits if you choose not to let your sample be used. However, once your sample has been studied and your genetic information has been put in the database, you will not be able to take that information back.

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HapMap  Re-­‐Consent  

The Repository does not let anyone sell material from samples or cell lines. However, information from genetics research sometimes helps companies make products to diagnose or treat diseases. If information from your family’s cell lines leads to making a product, it would probably contribute only in a very small way. Also, because the cell lines will not have names on them, neither the researchers nor anyone at the Repository would know if your samples were even used. So you will not get any additional payment for having your sample used in this project.

HapMap  Re-­‐Consent  

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… The database will not include any medical information about anyone whose sample is used. It also will not include any information that could identify who the individual people or families are. … Because the database will be public, people who do identity testing, such as for paternity testing or law enforcement, may also use the samples, the database, and the HapMap, to do general research. However, it will be very hard for anyone to learn anything about you personally from any of this research because none of the samples, the database, or the HapMap will include your name or any other information that could identify you or your family. 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 put in 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 if they thought your information might be in the database, got another sample from you, did many tests on that sample, and then compared the genetic information from those tests with the information in the database. The other is if somebody compared the information in the database with genetic information known to be from you that was in another database and figured out who you were. The risk of either of these things happening is very small, but it may grow in the future. We cannot always predict the results of research, so new risks to you may come up in the future that we can’t predict now.