Next	  Genera*on	  Sequencing:	  Reference	  Material	  (RM)	  Selec*on	  and	                   Design	                  ...
RM	  Scope	  •  Human	  Genome	  &	  Tumor	  Sequencing	  •  Variant	  Types	     –  SNP	     –  InDel	  /	  Subs*tu*on	  ...
Reference	  Material	  Needed	  For	  •  PlaVorm	  valida*on	      –  Sequencing	  System	      –  Bioinforma*cs/Analysis	...
New	  York	  State	  proposed	  dra[	  guidelines	  for	  NGS	  clinical	  cancer	  tests	  	  •  …	  A	  posi%ve/sensi%vi...
Associa*on	  for	  Molecular	  Pathology	      Comments	  to	  FDA	  UHT-­‐Sequencing	  Mee*ng,	  June	  2011	  •  …	  Per...
Reference	  Material	  PorVolio	  Whole	  Human	  Genome	  •  What	  sources	  of	  RMs	  to	  consider	        –  Availab...
Workgroup	  AAendees	  •  Approximately	  25	  aAendees	     –  Federal,	  incl.	  FDA,	  CDA,	  NIST	     –  Lab	  accred...
Workgroup	  Summary	  (1)	                  Reference	  Material	  Use	  •  Characterize	  PlaVorms	  &	  Methods	  	     ...
Workgroup	  Summary	  (2)	                    Selected	  Sample	  •  NA12878	  	      –  6,000	  controlled	  aliquots	  o...
Workgroup	  Summary	  (2)	                    Selected	  Sample	  •  NA12878	  	      –  6,000	  controlled	  aliquots	  o...
Workgroup	  Summary	  (3)	                                      Consent	  •  Consent	  available	  for	  	      –  Researc...
Workgroup	  Summary	  (4)	                  Sample	  &	  Consent	  •  If	  re-­‐consen*ng	  of	  NA12878	  not	  possible,...
Workgroup	  Summary	  (5)	                 Sample	  Characteris*cs	  •  Sample	  characteris*cs	  are	  more	  important	 ...
Workgroup	  Summary	  (5)	                         Sample	  Characteris*cs	  (cont.)	  •  High	  Priority	        –  Mul*p...
Workgroup	  Summary	  (6)	                     DNA	  RM	  Requirements	  •  DNA	  from	  low	  passage	  cell	  lines	    ...
Workgroup	  Summary	  (7)	                           Sample	  Source	  Sugges*ons	  Most	  support	  •  Personal	  Genome	...
Reference	  Material	  PorVolio	  Synthe*c	  DNA	  •  What	  types	  are	  most	  useful	       –  Variant	  types,	  sequ...
Reference	  Material	  PorVolio	                                                                           n ceSynthe*c	  ...
Confidential – Do not copy or distribute   | 19
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Reference Materials Selection and Design Working Group Summary Aug2012

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Reference Materials Selection and Design Working Group Summary Aug2012

  1. 1. Next  Genera*on  Sequencing:  Reference  Material  (RM)  Selec*on  and   Design   Workgroup   Genome  in  a  BoAle  Consor*um  /  NIST   August  16-­‐17,  2012  
  2. 2. RM  Scope  •  Human  Genome  &  Tumor  Sequencing  •  Variant  Types   –  SNP   –  InDel  /  Subs*tu*on   –  CNV   –  Structural  variant   | 2
  3. 3. Reference  Material  Needed  For  •  PlaVorm  valida*on   –  Sequencing  System   –  Bioinforma*cs/Analysis  Pipeline  •  Test  valida*on   –  Whole  genome   –  Targeted   –  Germline  vs.  tumor  •  Reagent  valida*on   | 3
  4. 4. New  York  State  proposed  dra[  guidelines  for  NGS  clinical  cancer  tests    •  …  A  posi%ve/sensi%vity  control  should  be  included  to   verify  analy*c  sensi*vity.  We  suggest  including  an   individually  bar  coded  low  posi*ve  control  near  the   sensi*vity  of  the  assay,  containing  mul*ple  known  soma*c   altera*ons  of  each  kind  to  be  detected,  to  verify  that  low   percentage  gene*c  variants  can  be  detected  in  every  run.  …  •  Performance  characteris*cs  for  each  type  of  gene*c   altera*on  the  assay  is  intended  to  detect,  e.g.  SNVs,  indels,   CNVs,  must  be  established  and  validated  •  Sequence  a  well-­‐characterized  reference  sample  (e.g.   HapMap  DNA  GM12878)  to  determine  error  rate  and   establish  depth/uniformity  of  coverage  across  all   amplicons.   | 4
  5. 5. 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  …  and  the  synthe*c  ERCC   RNA  transcripts  from  NIST  are  examples  of  poten%al   standard  reference  materials.  …   | 5
  6. 6. Reference  Material  PorVolio  Whole  Human  Genome  •  What  sources  of  RMs  to  consider   –  Available  for  research  and  for  profit   –  Primary/  cell  line  •  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   | 6
  7. 7. Workgroup  AAendees  •  Approximately  25  aAendees   –  Federal,  incl.  FDA,  CDA,  NIST   –  Lab  accredita*on   –  Clinical  reference  labs   –  PlaVorm  technologies   –  Reference  material  /  reagent  providers   –  Research   | 7
  8. 8. Workgroup  Summary  (1)   Reference  Material  Use  •  Characterize  PlaVorms  &  Methods     –  DNA  sequencing   –  Exis*ng  &  upcoming  NGS  technologies   –  Research  applica*ons   –  PlaVorm  &  methods  characteriza*on  for  clinical   diagnos*cs  applica*ons  •  Not  intended  as  reference  material  for   –  Valida*on  of  specific  muta*ons  in  a  panels   Confidential – Do not copy or distribute | 8
  9. 9. Workgroup  Summary  (2)   Selected  Sample  •  NA12878     –  6,000  controlled  aliquots  of  10ug  each  on  order  by   NIST  from  Coriell   | 9
  10. 10. Workgroup  Summary  (2)   Selected  Sample  •  NA12878     –  6,000  controlled  aliquots  of  10ug  each  on  order  by   NIST  from  Coriell  HOWEVER …. | 10
  11. 11. Workgroup  Summary  (3)   Consent  •  Consent  available  for     –  Research    ü  •  Consent  not  available  for   –  Commercial   •  Incl.  altera*ons,  re-­‐distribu*on   –  Re-­‐iden*fica*on  through  sequence  data   NA12878 and family requires re-consenting for commercial use and potential re-identification through sequence data | 11
  12. 12. Workgroup  Summary  (4)   Sample  &  Consent  •  If  re-­‐consen*ng  of  NA12878  not  possible,   replace  with  other  sample  (Trio)  •  If  necessary,  do  sample  change  now,  since   early  in  process  of  selec*ng  reference  sample  •  New  characteriza*on  of  reference  material   will  be  in  much  greater  depth  and  with   technologies  that  are  more  advanced  than   exis*ng  data  for  NA12878   | 12
  13. 13. Workgroup  Summary  (5)   Sample  Characteris*cs  •  Sample  characteris*cs  are  more  important   than  selec*on  of  specific  sample  IDs  •  More  reference  samples  preferred  over  fewer   samples   –  Prefer  8  fully  characterized  samples  at  high  depth   and  corresponding  trios  at  lower  depth  over  4   fully  characterized  samples  plus  trios   | 13
  14. 14. Workgroup  Summary  (5)   Sample  Characteris*cs  (cont.)  •  High  Priority   –  Mul*ple  ethnici*es   •  Diversity  in  structural  varia*on  to  stress  systems   –  Balanced  female  to  male  ra*o   –  Cell  line,  low  passages   •  Replenish  supply  •  Nice  to  have   –  Interracial  marriage  samples     •  Controlled  admixture  •  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. Workgroup  Summary  (6)   DNA  RM  Requirements  •  DNA  from  low  passage  cell  lines   –  New  batches  from  low  passage  aliquots  •  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  dri[,  mix  ups,  ….  ,  develop  benchmarks  •  Reference  material  that  mimics  tumor  sample  characteris*cs   –  FFPE  embedded  cells?   | 15
  16. 16. Workgroup  Summary  (7)   Sample  Source  Sugges*ons  Most  support  •  Personal  Genome  Project  Samples     –  Includes  trios     –  Use  sequence  data  to  derive  admixture   –  hAp://www.personalgenomes.org   –  Consent  includes  research  &  commercial  use  and  re-­‐iden*fica*on    Some  support  (if  consent  sufficient)  •  HS1011   –  Charcot  Marie  Tooth  cell  line   •  Lupski  et  al,  NEJM  2010  •  MCF10A     –  Normal  breast  cancer   •  Used  by  Horizon  Dx  to  produce  isogenic  cell  lines  with  cancer  relevant  muta*ons  Other  •  African  American  sample  with  70%  sanger  sequence  (ID?)   –  No  cell  line  available   –  Subject  s*ll  alive  =>  re-­‐consent  &  generate  cell  line?   | 16
  17. 17. Reference  Material  PorVolio  Synthe*c  DNA  •  What  types  are  most  useful   –  Variant  types,  sequence  context,  phasing   •  Assess  strengths  &  weaknesses  of  plaVorm   •  Test  valida*on  for  exis*ng  and  medically  relevant  variants  •  What  size  •  How  many  synthe*c  RMs   –  PlaVorm  valida*on   –  Test  valida*on  •  Which  exis*ng  sources  should  be  considered   –  Available  for  research  and  for  profit  •  Source  of  commercial  development  and  distribu*on   –  Manufactured  under  quality  system  for  diagnos*c  applica*ons   | 17
  18. 18. Reference  Material  PorVolio   n ceSynthe*c  DNA  •  What  types  are  most  useful   –  Variant  types,  sequence  context,  phasing   n -co fere e by tele artic ipat •  Assess  strengths  &  weaknesses  of  plaVorm   What  size   vie w to p •  Test  valida*on  for  exis*ng  and  medically  relevant  variants  •  Resynthe*c  Rnvited iMs   are•  How  many   ll Aalida*on   –  PlaVorm  valida*on   –  Test  v•  Which  exis*ng  sources  should  be  considered   –  Available  for  research  and  for  profit  •  Source  of  commercial  development  and  distribu*on   –  Manufactured  under  quality  system  for  diagnos*c  applica*ons   | 18
  19. 19. Confidential – Do not copy or distribute | 19
  20. 20. Divider slide

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