Vienna    16/05/2017  

Presented  by  Antonio  Alonso  on  behalf  of  the  DNASEQEX  ConsorAum    

61%  18%  

12%  6%   3%  

Type  of  Respondent  Organiza3ons  

Police  laboratories   Ministry  of  JusAce   University  

Ministry  of  Health   Private  Agency  

33  ENFSI  DNA  WG  laboratories  from  25  countries  

ParAcipaAon:  54%  of  all  ENFSI  DNA  Labs  (53  Member  InsAtutes  and  8  Associated  Members)  

32  

30  

30  

23  

29  

5  

0   5   10   15   20   25   30  

Forensic  DNA  analysis  from  traces  in  criminal  cases  

UnidenAfied  Human  Remains  and  Missing  Persons  

ContribuAon  To  NaAonal  DNA  Databases    

Parentage  TesAng    

Research  and  ValidaAon  

Others  

Number  of  Labs    

Indicate  the  areas  of  forensic  DNA  applica3ons  performed  in  your  laboratory  

52%  48%  

Does  your  laboratory  have  a  Massively  Parallel  Sequencing  instrument?  

YES  

NO  61%  

39%  

Does  your  laboratory  have  a  Massively  Parallel  

Sequencing  instrument  or  Will  your  laboratory  purchase  a  MPS  instrument  in  the  next  

18  months?  

Yes  

No  

3.6  3.2  3  

2.7  2.7  

1.95  1.9  1.9  

0   1   2   3   4   5  

No  available  funding  

Methodology  is  too  expensive  

WaiAng  unAl  MPS  applicaAons  are  more  fully  developed  

Instruments  are  too  expensive  

No  experAse  available  to  perform  bioinformaAcs  

No  experAse  available  to  perform  tesAng  

No  lab  space  available  to  perform  tesAng  

No  need  for  the  technology  (CE  addresses  the  vast  majority  

What  are  the  reasons  for  currently  NOT  purchasing  a  MPS  instrument?    

Responses  from  16  labs  ranked  in  order  of  priority  from  1(lowest)  to  5  (highest)  

41%  

41%  

18%  

Please  indicate  the  methods  used  for  DNA  library  prepara3on,  DNA  template  

prepara3on,  and  cloning  

Manual   Manual  and  Automated   Automated  

10  

7  

4  3  

How  many  of  the  following  MPS  instruments  does  your  laboratory  have?  

Illumina  MiSeq  

Ion  Torrent  PGM  

Ion  Torrent  S5  

Others  (1  lab)  

2  Hamilton  &  1  Beckman  placorms  

for  Illumina  Workflow  

7  Ion  Chef™    for  Thermofisher  Workflow  

AutomaAon  (somehow)  leads  us  away  from  scienAfic  knowledge  •  User   guides   on   automated   MPS   Technology   should   be   comprehensive,   describing   in   detail   the   different  

processes  and  steps  involved  in  DNA  library  and  DNA  template  preparaAon  (Genomic  PCR,  Barcoding  by  ligaAon  or  PCR,  Library  equalizaAon  and  purificaAon,  Solid-­‐PCR  of  libraries,  Enrichment,  Chip  Loading,..).    

•  Informa3on  on  relevant  parameters  (such  as  PCR-­‐Setup  and  pipelng  parameters,  PCR  parameters,  PurificaAon  parameters,…)  should  also  be  included  as  it  is  needed  for  the  validaAon  and  the  interpretaAon  of  the  data.  

10  

9  

2  

6  

0   2   4   6   8   10   12  

Illumina  UAS  

Torrent  Suite  Sonware  

Converge  (Beta  version)  

Open  Source  Sonware    

Please  indicate  the  soYware  used  for  Sequence  Assembly  and  Data  Analysis  

Open  Source  Sonware  

•  STRait  Razor  v2.0    •  FDSTools  

72%  

14%  14%  

How  do  you  store  MPS  data  generated  in  your  laboratory?  

In-­‐house  server  

Instrument  Manufacturer  Owned  Cloud  

External  Server    

DNASeqEx  Sonware  ValidaAon  

Data  analysis  of  MPS-­‐STR  panels  by  Commercial  SoYware    (UAS  and  TSS/Converge)  

Improvements  to  recover  full  informa3on  from    MPS  sequence  data      

•  Sequence  coverage  data  on  forward  and  reverse  strands  

•  STR  array  sequence  data  +  flanking  regions  sequence  data  

•  Genome  coordinates  of  the  sequence  read  start  and  end  points  

•  Comprehensive  and  global  STR  nomenclature  system  (backward  compaAbility  with  STR  CE  data)  

•  Global  output  file  for  internaAonal  exchange    

14  

11  9  

11  

8   8   8  

3   3  2  

19  18  

17  15   15  

14  

11  

8   8  

3  

Please  check  for  which  category  or  categories  of  Forensic  DNA  markers  MPS  is  currently  used  or  being  evaluated  in  your  lab  and  

for  which  ones  will  be  developed  in  the  next  18  months  

Number  of  labs  in  December  2016   Number  of  labs  in  2018  

>  75%    

Innsbruck     Berlin     Madrid  

DNASeqEx  ValidaAon  studies  

STR  Kits   Valida3on  Studies  

•  ForenSeq™  DNA  Signature  Prep  Kit    (Panel  A:  27  Autosomal  STR,  24  Y-­‐STR,  7  X-­‐STR,  and  94    SNPS)  •  Precision  ID  GlobalFiler®  Mixture  ID  Panel  (29  autosomal  STRs,  1  Y  STR,  42  SNPs,  2  Y-­‐  SNPs,36  microhaplotypes  clusters,  Amelogenin  and  the  Y  indel  rs2032678)  •  Precision  ID  GlobalFiler®  STR  ID  Panel  (29  autosomal  STRs,  1  Y-­‐STR,  Amelogenin)  

•  AnalyAcal,  InterpretaAon  and  Stuter  Thresholds  

•  Concordance  to  CE  •  Reproducibility    •  SensiAvity    •  Mixture  studies  •  PopulaAon  studies    •  Forensic  samples  

DNASeqEx  ValidaAon  studies  

STR  Kits   Mee3ng  Presenta3on  

•  ForenSeq™  DNA  Signature  Prep  Kit    (Panel  A:  27  Autosomal  STR,  24  Y-­‐STR,  7  X-­‐STR,  and  94    SNPS)  

STR  Kits   Mee3ng  Presenta3on  

•  Precision  ID  GlobalFiler®  Mixture  ID  Panel  

(29  autosomal  STRs,  1  Y  STR,  42  SNPs,  2  Y-­‐  SNPs,36  microhaplotypes  clusters,  Amelogenin  and  the  Y  indel  rs2032678)  •  Precision  ID  GlobalFiler®  STR  ID  Panel  (29  autosomal  STRs,  1  Y-­‐STR,    Amelogenin)   Pedro  Barrio  

Petra  Müller  Do  not  miss  it.    

Today  15:20–16:20  

3.7  

3.5  

3.4  

3.1  

2.8  

2.3  

2  

0.4  

0   0.5   1   1.5   2   2.5   3   3.5   4  

Lack  of  nomenclature  and  reporAng  standards      

NaAonal  DNA  Databases  cannot  accommodate  the  new  data  

Not  sufficient  populaAon  data  

There  is  a  lack  of  an  adequate  naAonal  legislaAon  framework  

No  specific  validaAon  guidelines  available    

No  specific  proficiency  tests  available  

Not  demonstrated  to  be  reliable  

Others  

What  do  you  think  are  the  main  scien3fic  and  legal  challenges  for  the  implementa3on  of  MPS  in  Forensic  Gene3cs?  

Responses  from  33  labs  ranked  in  order  of  priority  from  1(lowest)  to  5  (highest)  

Nomenclature  and  Na3onal  DNA  Databases    compa3bility      

ü  Simple   STR   nomenclature   systems   (numbers   and   le-ers)     are   easy   to  communicate   and   therefore   preferred   for   rou7ne   exchange   of   STR   data   between  analysts  and  stakeholders  

ü  However,   the   transla7on   process   will   have   to   be   managed   by   a   centralized  nomenclature  commission   to  avoid  ambiguous  or   imprecise  allele  names  being  adopted,  or  assigning  different  names  to  iden7cal  alleles.  

ü  It  has  been  suggested  that  an  online  system  could  be  used  that  is  curated  by  a  nomenclature   commission,   which   would   be   responsible   for   new   allele  designa7ons  upon  valida7on  of  the  observed  sequence  varia7on.  

ü  Criteria  for  the  valida:on  of  sequence  varia:on  and  its  comparison  with  exis7ng  variants  need  to  be  defined  in  more  detail  

Popula3on  studies  on  STR  sequence  varia3on  

A  publicly  available,  centrally  curated  online  allele  frequency  database  and  

quality  control  plaHorm  

STR  sequence  data  in  STRidER        

The   storage   of   nucleo:de   strings   (text   strings)   in   FASTA-­‐like   format   has  been   iden7fied   as   the   core   framework   to   permit   soIware-­‐aided   alignment   and  transla7on  between  STR  nomenclatures  (MPS  &  CE).      The   exis7ng   architecture   of   STRidER   allows   for   the   implementa7on   of   nucleo7de  sequence  strings  (as  kept   in  EMPOP)  and  thus   is  fully  compa:ble  with  the  QC  of  popula:on  data  generated  by  MPS.  

DNASeqEx  Present  and  Future  Plans  

SYSTEMATIC  EVALUATION  OF  MASSIVELY  PARALLEL  STR  SEQUENCING  DATA  MiSeq  FGx  benchtop  sequencer  (Illumina  Inc.,  CA)  or  the  Ion  S5  sequencing  system  (Thermo  Fisher  ScienAfic  Inc.,  CA)      LIMS  INTEGRATION  OF  NEW  MASSIVELY  PARALLEL  SEQUENCING  WORKFLOWS  IN  FORENSIC  GENETICS      ConfiguraAon  of  a  commercial  LIMS  system  (LabWare  Inc.,  DE)  to  fit  the  automated  workflow  provided  by  the  Ion  Chef  /  Ion  Torrent  S5XL  placorms  and  the  Torrent  Server  Suite  /  Converge  sonware  packages.      NOMAUT  –  NGS  STR  Nomenclature  for  Forensic  Gene3cs  Web  Self-­‐maintaining  NOMenclature  AUThority  sonware  

 Acknowledgement    

ParAcipaAng  Laboratories  

 The                                                          Faculty  

InsAtute  of  Legal  Medicine,  Medical  University  of  Innsbruck.  Austria  

InsAtute  of  Legal  Medicine  and  Forensic  Sciences,    Charité  –Universitätsmedizin  Berlin.  Germany    

Center  for  Human  IdenAficaAon  at  the  University  of    North  Texas  Health  Science  Center.  EEUU    

NaAonal  of  Toxicology  and  Forensic  Sciences.    Madrid  Department.  Spain    

Steffi  Köcher  

Speaker   was   provided   travel   and   hotel   support   by   Thermo   Fisher   ScienAfic   for   this   presentaAon,   but   no  remuneraAon    When  used  for  purposes  other  than  Human  IdenAficaAon  or  Paternity  TesAng  the  instruments  and  sonware  modules  cited  are  for  Research  Use  Only.  Not  for  use  in  diagnosAc  procedures.    Thermo   Fisher   ScienAfic   and   its   affiliates   are   not   endorsing,   recommending,   or   promoAng   any   use   or  applicaAon  of  Thermo  Fisher  ScienAfic  products  presented  by  third  parAes  during  this  seminar.   InformaAon  and  materials   presented  or   provided  by   third  parAes   are  provided   as-­‐is   and  without  warranty  of   any   kind,  including   regarding   intellectual   property   rights   and   reported   results.   ParAes   presenAng   images,   text   and  material  represent  they  have  the  rights  to  do  so.  

 Acknowledgements    

This   project   has   been   funded   with   support   from   the   European   Commission   (grant  HOME/2014/ISFP/AG/LAWX/4000007135   under   the   Internal   Security   Funding   Police  programme   of   the   European   Commission-­‐Directorate   General   JusAce   and   Home  Affairs).   This   publicaAon   reflects   the   views   only   of   the   authors,   and   the   European  Commission   cannot   be   held   responsible   for   any   use   which   may   be   made   of   the  informaAon  contained  therein.