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Interview with Manfred Kayser - DNA Intelligence

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An Interview with Professor Manfred Kayser on Next Generation Sequencing with the Ion PGM™ Semiconductor sequencer: A new era of DNA Intelligence. …

An Interview with Professor Manfred Kayser on Next Generation Sequencing with the Ion PGM™ Semiconductor sequencer: A new era of DNA Intelligence.

Professor Manfred Kayser is the founding head of the Department of Forensic Molecular Biology at the Erasmus University Medical Center in Rotterdam, the Netherlands.

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  • 1. Next  Generation  Sequencing  with  the  Ion  PGM™  Semiconductor  sequencer:  A  new  era  of  DNA   Intelligence.   Professor   Manfred   Kayser   is   the   founding   head   of   the   Department   of   Forensic   Molecular   Biology   at   the   Erasmus   University   Medical   Center   in   Rotterdam,   the   Netherlands.   Grounded   in   science   and   driven   by   curiosity,   his   major   motivation   is   to   develop   practical   applications   and   provide   solutions   to   currently   unsolvable   questions   in   forensics,   in   areas   where   genetics   and   molecular   biology  can  be  of  help.    Fascinated  by   anthropology   and   the   genetic   history   of   human   populations,   one   of   Manfred   Kayser’s   research   focuses   is   on   bio-­‐geographic   ancestry   and  understanding  why  genetically  we  look  the  way  we  look.       Life  Technologies  spoke  to  Professor  Manfred  Kayser  about  next  -­‐generation  sequencing  (NGS)  for   forensic  applications.       There   is   a   lot   of   discussion   about   NGS   within   the   forensic   community.   Both   routine   labs,   and   in   particular,  specialised  research  labs  are  interested  in  this  technology  and  want  to  understand  how   NGS  will  impact  Forensics.     The   biggest   impact   is   that   we   will   be   able   to   combine   a   large   number   of   genetic   markers   of   different  types  in  a  single  run  analysis  including  markers  such  as  STRs  and  SNPs  (single  base  pair   changes),   potentially   even   RNA   that   cannot   be   multiplexed   with   any   other   current   technology.   This   will   open-­‐up   new   possibilities   as   various   types   of   forensically   relevant   information   can   be   obtained  from  a  single  sample  in  a  single  analysis.     Of  particular  interest  here  is  the  use  of  SNPs  for  Forensic   DNA  Phenotyping  (FDP),  which  includes  DNA  markers  to   My  postdocs,  PhD  students   predict   externally   visible   characteristics   and   DNA   and  technicians  are   markers   to   infer   bio-­‐geographic   ancestry   and   can   help   in   enthusiastic  about  the   finding   missing   persons   or   perpetrators   for   whom   the   machine.  For  most  projects,   STR  profile  is  not  already  known  to  the  authorities.  This   set  up,  implementation  and   will  provide  useful  information  for  the  police  to  be  used   as   investigative   leads   when   searching   for   unknown   getting  results  –  it  worked   and  it  worked  very  quickly.    
  • 2. perpetrators,  but  can  also  be  useful  in  missing  person  identification.     Of   interest   may   also   be   SNPs   for   individual   identification   as   they   provide   strong   technical   advantages  over  STRs,  such  as  no  slippage  artifacts  and  better  ability  to  deal  with  degraded  DNA   due   to   smaller   fragment   length.   However,   their   implementation   in   practical   routine   is   troubled   by   the  fact  that  existing  forensic  DNA  (profile)  databases  are  all  STR-­‐based.       NGS   analysis   of   forensic   STRs   also   has   advantages   as   it   will   reveal   microvariations   providing   additional   resolution,   but   because   these   are   not   covered   with   conventional   fragment   length   analysis   they   are   not   (yet)   included   in   the   existing   forensic   DNA   databases   and   thus   of   limited   use   until  this  information  gets  included  in  the  databases.  Furthermore,  NGS  analysis  of  forensic  STRs   allows  a  more  direct  and  thus  more  accurate  quantification  of  PCR  products,  which  is  of  help  in   mixture  interpretation.  However,  when  it  simply  comes  to  STRs  the  problem  of  slippage  artifacts   and   their   impact   on   mixture   interpretation   remains   also   with   NGS   platforms   as   long   as   they   are   PCR-­‐based.         Do  you  think  NGS  will  transform  forensics?   Moving  to  a  new  technology  is  not  easy  in  forensics,  which  traditionally  is,  and  in  a  way,  has  to  be   a  conservative  field.  Change  takes  time.  NGS  if  implemented  into  forensics  will  change  the  whole   workflow  from  sample  preparation  to  data  interpretation  and  storage,  and  every  step  needs  to  be   validated.   Specialised   forensic   labs,   and   dedicated   companies,   are   already   working   to   develop   robust   protocols   and   to   validate   their   methods.   Some   labs,   including   ours,   are   working   on   SNP   panels,  others  concentrate  on  mtDNA  sequencing  protocols,  or  on  forensic  STR  sequencing.       The  main  issues  for  forensics  will  be:  can  the  forensic  community  be  convinced  to  change  to  NGS   and  also  can  the  evidence  generated  by  NGS  stand  up  in  court?     NGS   generates   a   different   kind   of   data   where   you   use   sequencing   to   e.g.   directly   estimate   the   number  of  STR  repeats  instead  of  assuming  repeat  counts  from  fragment  length  analysis  as  done   with   conventional   STR   analysis.   This   is   more   objective   and   allows   better   quantification,   but   acceptance  of  this  as  evidence  in  court  will  take  time.     NGS   generates   more   information   than   conventional   CE   based   analysis.   More   markers,   more   samples,   more   results   from   low   amounts   of   (degraded)   DNA.   That   is   a   real   benefit   and   may   motivate   forensic   DNA   analysts   to   consider   changing   their   workflow.   But   it   is   essential   to   communicate  the  benefits  of  NGS  and  to  define  ‘more  information’.     Another  issue  of  course  is:  National  Forensic  DNA  Databases.  Huge  investments  have  been  made   to  set  up  the  current  STR  based  databases  and  this  may  be  used  as  an  argument  for  not  wanting  to   change   to   new   markers.   However,   it   is   important   to   realise   that   most   databases   will   become  
  • 3. obsolete  at  some  point  in  time  as  convicted  offenders  are  imprisoned,  ‘retire’,  or  die.  Now  it  may   be   a   good   time   to   establish   parallel   databases,   i.e.   based   on   SNPs   for   individual   identification,   and   introduce  new  marker  systems  to  the  database  while  maintaining  current  STR  profiles.  Especially   with   having   a   technology   such   as   NGS   available   that   can   analyse   SNPs   together   with   STRs   in   a   single  run  so  that  no  extra  evidence  material  is  spent  for  the  extra  analysis  of  the  new  markers.         What  will  be  the  benefits  of  NGS  for  routine  forensic  labs?   One   benefit   of   NGS   is   to   be   able   to   multiplex   not   only   a   large   number   of   genetic   markers   of   different   types   but   also   samples   by   using   barcodes   on   individual   DNAs.   This   strategy   is   widely   employed  outside  forensics  to  reduce  cost  per  sample  considerably  but  also  to  take  full  advantage   of  the  large  coverage  capacity  of  NGS  platforms.  However,  I  have  heard  reservations  from  forensic   colleagues   on   the   idea   of   mixing   forensic   evidence  even   though   it   is   scientifically   and   practically   possible  with  this  technology.     To  me,  the  biggest  advantage  is  the  combination  of  different  marker  types  for  different  forensic   purposes  also  beyond  individual  identification  such  as  for  Forensic  DNA  Phenotyping  and  others.       What  are  your  goals  for  implementing  NGS  in  your  research?       The   focus   in   my   department   is   the   combination   of   fundamental   and   applied   research.   I   want   to   drive   innovation   and   develop   new   science   and   technologies   suitable   for   forensic   requirements.   This   is   a   different   approach   to   what   mostly   happened   in   the   past   where   usually   forensic   people   waited   for   new   knowledge   to   be   developed   by   people   working   in   fundamental   sciences,   and   then   adopted   it   for   forensic   applications.   If   you   consider   that   many   important   discoveries,   including   multilocus   DNA   fingerprinting   are   done   more   or   less   by   chance,   it   is   clear   that   this   is   not   a   very   effective  strategy.     We   try   to   approach   things   more   directly.   Usually   we   take   an   open   question   in   forensic   routine,   carefully  check  if  molecular  genetics  or  molecular  biology  may  be  able  to  provide  a  solution,  and  if   so   perform   basic   research   in   such   forensically   relevant   area   of   molecular   biology   /   genetics   and   produce   new   scientific   knowledge.   If   the   gained   new   knowledge   is   sufficient   enough   to   address   the   forensic   question,   we   further   continue   to   make   this   new   knowledge   suitable   for   forensic   applications  by  developing  and  forensically  validating  new  test  systems  that  eventually  can  help  in   solving  the  previously  unsolved  forensic  problem.   Examples   are   our   activities   in   predicting   bio-­‐geographic   ancestry   and   externally   visible   characteristics   from   DNA.   We   already   have   found   genes   and   predictive   DNA   markers   and   developed   DNA   test   systems   for   eye   and   hair   colour   prediction   to   be   usable   in   forensic   labs   for   Forensic   DNA   Phenotyping   purposes,   and   markers   /   systems   for   more   appearance   traits   are  
  • 4. underway.   We   have   also   investigated   patterns   of   genome-­‐wide   gene   expression   for   developing   mRNA  and  miRNA  markers  and  RNA  test  systems  for  forensic  tissue  identification.     Furthermore,   we   previously   studied   the   mutability   of   large   numbers   of   STRs   from   the   non-­‐ recombining   part   of   the   Y   chromosome   and   identified   Y-­‐STRs   that   mutate   much   faster   than   the   ones  currently  used  in  forensics.  These  so  called  rapidly-­‐mutating  (RM)  Y-­‐STRs  provide  enhanced   suitability   to   differentiate   between   male   relatives   that   usually   with   the   currently   used   Y-­‐STRs   cannot  be  separated  marking  a  limitation  of  Y-­‐STRs  over  autosomal  STRs  for  human  identification,   which   partly   can   be   overcome   with   RM   Y-­‐STRs.   We   are   also   performing   research   in   circadian   biology  and  working  on  biomarkers  and  test  systems  that  allow  estimating  the  time  when  a  stain   was   placed   at   a   crime   scene,   a   kind   of   biological   alibi   test.   As   you   see,   we   are   active   in   developing   various  markers  of  different  types  for  various  forensic  purposes.     Why  did  you  choose  the  Ion  PGM™  Semiconductor  Sequencer?   We   were   interested   in   a   device   that   allows   combining   the   various   markers   we   have   developed   (and   will   further   do)   for   the   various   forensic   purposes   together   with   STRs   in   a   single   parallel   analysis,   and   for   that   the   Ion   PGMTM   System   seems   to   be   suitable.   Apart   from   all   technical   advantages  the  Ion  PGMTM  Sequencer  offers,  there  also  is  a  historic  reason!  My  relationship  with   Life   Technologies/Applied   Biosystems®   goes   back   for   quite   a   while   already.   Working   with   Life   Technologies  has  been  and  is  a  very  positive  experience.       How  easy  was  it  to  setup  the  Ion  PGMTM  Sequencer  in  your  lab?   The   setup   of   the   Ion   PGM™   System   and   getting   started   was   fairly   easy.   The   work   flow   is   straightforward   and   we   had   no   problems   with   implementing   the   Ion   PGMTM   Sequencer   in   our   research  lab  depending  a  bit  on  the  project  complexity.   For   some   projects,   such   as   massive   parallel   SNP   typing,   we   had   almost   no   problems   implementing  the  Ion  PGMTM  Sequencer.  Also,   RNA   marker   typing   with   the   Ion   PGMTM   System   provided   almost   no   problems   and   was   implemented  rather  quickly.   We   also   use   the   Ion   PGMTM   System   for   more   challenging   projects   such   as   forensically   motivated   transcriptome   sequencing   where   naturally   the   implementation   process   is   more   complex  and  thus  needs  more  time.  Also  data  handling  and  interpretation  is  more  challenging  in  
  • 5. such  projects  where  the  amount  of  data  produced  is  by  magnitudes  larger  than  when  genotype-­‐ by-­‐sequencing  a  few  hundreds  of  SNPs.     My  postdocs,  PhD  students  and  technicians  are  enthusiastic  about  the  machine.  For  most  projects,   set  up,  implementation  and  getting  results  –  it  worked  and  it  worked  very  quickly.     To   find   out   more   information   on   the   Ion   PGM™   Sequencer,   visit   our   website   at   http://www.lifetechnologies.com/PGM,   and   on   Manfred   Kayser   and   his   Department   of   Forensic   Molecular   Biology   at   Erasmus   MC   University   Medical   Center   Rotterdam,   visit   his   website   at   http://www.erasmusmc.nl/fmb.     Research,  Forensic  or  Paternity  Use  Only.  Not  for  use  in  diagnostic  procedures.     ©  2013  Life  Technologies  Corporation.  All  rights  reserved.        The  trademarks  mentioned  herein  are  the  property  of  Life  Technologies  Corporation  and/or  its  affiliate(s)  or  their   respective  owners.