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Next genera*on mtGenome sequencing for forensic purposes using the Ion Torrent PGM Dr. Walther Parson Ins*tute of Legal Medicine, Innsbruck Medical University, Austria [email protected] www.empop.org Ion Torrent™ Semiconductor Sequencing for Forensic Applications Webinar Feb 14, 2013

Next generation mtGenome sequencing for forensic purposes using the Ion Torrent PGM™ – Dr. Walther Parsons

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Next  genera*on  mtGenome  sequencing  for  forensic  purposes  using  the  Ion  Torrent  PGM  

 Dr.  Walther  Parson    

Ins*tute  of  Legal  Medicine,  Innsbruck  Medical  University,  Austria  walther.parson@i-­‐med.ac.at  

www.empop.org  

Ion Torrent™ Semiconductor Sequencing for Forensic Applications Webinar Feb 14, 2013

Mitochondrial  DNA  

 circular  double-­‐stranded  molecule    coding  region  (15  kb)  37  genes    control  region  (1.1  kb)  d-­‐loop  

 evolu?onary  rate  ~10x  of  nDNA  

(www.mitochondrialdnates?ng.com)  

Russell,  P.  J.  (2005)

High  mtDNA  copy  number  

Higher  copy  number  than  nDNA    4-­‐5  mtDNA  (avg)  molecules/mitochondrion  (Satoh  and  Kuroiwa,  1991)    up  to  1,000  mitochondria/cell  (Robin  and  Wong,  1988)  

Applica*on  to  challenging  crime  stains  (e.g.  hair)  

Szabo et al 2012

MtDNA  is  maternally  inherited  

Mitochondria  derive  from  the  fer?lized  egg  –  passed  along  maternal  line  

(www.accessexcellence.org)

(anthro.palomar.edu)

Applica*on  to  old  historical  cases  

fibula (Günter Messner) Parson et al 2007

femur (Romanov family) Coble et al 2009

premolar (Wolfgang A. Mozart) Parson 2006

The Genographic Project

Phylogeny  of  haploid  DNA  markers  

Why  mtGenomes?  

discrimina?on  power,  phylogeny,  quality  control    

2011-­‐MU-­‐MU-­‐K402  collabora?on  with  AFDIL  (Dover,  DE)    

Maximizing  mtDNA  Tes?ng  Poten?al  with  the  Genera?on    of  High-­‐Quality  mtGenome  Reference  Data  

Amplicon-­‐based  sequencing  more  sensi?ve  than  compe?tor  instruments    Amenable  to  degraded  DNA  (down  to  75  bp)    Mul?plex  individuals  –  Barcodes  (up  to  96)    Faster  library  construc?on    Automated  enrichment  system    Very  fast  sequencing  ?me    Less  total  hands-­‐on  ?me    Higher  throughput  (runs  per  week)    Natural  chemistry  (less  bias,  less  maintenance)  

Why  PGM?  

42  mtGenomes  sequenced  with  STS  and  PGM  

Samples  

Origin   #   Source   Reference  

Sub-­‐Saharan  (Angola)   5   blood   Fendt  et  al  2012  

Southeast  Asian  (East  Timor)   8   buccal   this  study  

Westeurasian  (Austria)   6   paraffin-­‐embedded  ?ssue   Fendt  et  al  2011  

Westeurasian  (Austria)   23   buccal   this  study  

Sanger-­‐type  Sequencing  of  mtGenomes  

mtGenome  PCR  with  2  and  9    overlapping  amplicons

Sequencing  using  106  primers

or

OneTouch™   OneTouch™ES   PGM™   Torrent  Server  &    Torrent  Browser  courtesy Applied Biosystems by Life technologies

PCR   e-­‐shearing  (130-­‐140  bp)  

100/200  bp  chemistry  316  chips  

PGM  Next  Genera*on  Sequencing  of  mtGenomes  

BAM  (Binary  Alignment  Map),  BAI  (Binary  Alignment  Index)    rCRS  (mtDNA  Reference  sequence,  Andrews  et  al  1999)    Torrent  Browser  Variant  Caller  (Ion  Torrent,  Life  Technologies)  

 Variant  Caller  (Vs.  3.2.43647)    TMAP  Smith-­‐Waterman  alignment  op?miza?on  (Li  and  Homer,  2010)  

 Integra?ve  Genomics  Viewer  (IGV)  

 Freeware  to  visualize  alignment  files  (Robinson  et  al,  2011;  ,  Vs.  2.1.21  (2541  )    paired  read  alignment  

 NextGENe  (SokGene?cs)  

 paired  read  alignment  (Vs.  2.3.1)  +  visualiza?on    Sequencher  (GeneCodes)  

 Tablet  for  NGS  integrated  in  Sequencher  (5.0)    GSNAP  alignment  (Wu  and  Nacu,  2010)  

 

NGS  analysis  tools  used  in  this  study  

Results  

Loading  density  on  316  chip  

Ion  Torrent  Variant  Caller  

20%  variant  frequency  threshold  (of  total  coverage)  because  some  low  DNA  templates  were  used  for  NGS  

Other  alignment  methods  with  viewers  

Direct  comparison  between  STS  and  PGM  

PGM  seq.  chem.     #   bp   differences  

100  bp     31   513,651     95  (0.018%)*  

200  bp   33   546,786     81  (0.015%)*  

16183/4' 16193.XC' 309.XC' 315.1C' 573.XC' Del' Sub'

16183/4'

16193.XC'

309.XC'

315.1C'

573.XC'

Del'

Sub'

16183/4' 16193.XC' 309.XC' 315.1C' 573.XC' Del' Sub'

100  bp 200  bp

* Length heteroplasmy not considered; alignment 5’ (PGM) vs. 3‘ (STS)

HVS-­‐2  C  tract  -­‐  315.1C  

PGM   #  

100  bp     31  (100%)  

200  bp   33  (100%)  

6C  T310   G316  

Seems  to  be  an  artefact  of  the  TMAP  aligner    If  a  modified  rCRS  with  6Cs  between  310  and  316  is  used  as  reference,  all  6  C’s  are  reported  by  the  Ion  Torrent  variant  caller    Paired  read  aligner  report  the  insCs  rela?ve  to  rCRS  (see  later)  

100  bp   200  bp  

(rCRS  variant) 5C  

HVS-­‐2  C  tract  -­‐  309.XC  

PGM   #  

100  bp     16  (51.6%)  

200  bp   14  (42.4%)  

7C   T310  

All  C-­‐tracts  between  302  and  310  with  the  rCRS  variant  (7  Cs)  were  concordantly  reported  between  STS  and  PGM    Inser?ons  of  1  and  2  Cs  (309.1C,  309.2C)  were  not  reported  with  PGM  (TMAP  alignment  artefact)  

100  bp   200  bp  

rCRS  variant

HVS-­‐1  C  tract  -­‐  16193.XC  

PGM   #  

100  bp     3  (9.7%)  

200  bp   5  (15.2%)  

4C  

T16189  

100  bp   200  bp  

5C  

16183C  16189C  16193.1C  

rCRS  variant

extensive  LHP All  rCRS  variants  concordant

Effect  of  different  aligners  on  C  tract  reports  

Sanger NextGene - mutation report position score cv 309.1C 310 insC 6,1 130 315.1C 316 insC 2,9 123 16189C 16189 T>C 15,9 208 16193.1C - - -

WGS28  

NextGENe   IGV   Sequencher  Tablet  

Subs*tu*ons  

PGM   #  

100  bp     29  

200  bp   15  100  bp   200  bp  

Twenty-­‐two  (75.9%)  differences  in  100  bp  chemistry  caused  by  6  samples  (not  sequenced  with  200  bp  chemistry  due  to  low  DNA  amount).      Gave  also  low  EPGs  for  STS.        

Majority  of  subs?tu?on  differences  in  200  bp  chemistry.  Some  posi?ons  were  hit  in  mul?ple  samples:  10651  (3),  10664  (3),  2689  (3)    lie  close  to  PCR  primer  binding  regions    456  (4)    short  T-­‐stretch  between  452-­‐455  (alignment)    

Dele*ons  (outside  HVS-­‐tracts)  

PGM   #  

100  bp     11  

200  bp   13  100  bp   200  bp  

A  total  of  13  (54.2%)  reported  dele?ons  observed  together  with  a  subs?tu?on  at  the  same  posi?on  (the  laser  confirmed  by  STS)    e.g.  WGS05  hg  L0d1’2  498del  not  reported  for  200  bp  chemistry,  present  in  IGV  viewer  as  494del  

494del

Substan?ally  higher  through-­‐put  of  mtGenome  sequencing  with  PGM  compared  to  STS    High  concordance  between  STS  and  PGM  reports  for  both  chemistry  versions  (176  differences  in  1,060,437  bps  -­‐  0.017%;  20%  variance  call  threshold)    Roughly  two  thirds  of  differences  observed  in  homopolymeric  C  tracts  (>  7Cs);  physical  PGM  sequence  data  seem  to  be  there,  as  modified  rCRS  or  other  alignment  algorithms  reveal  concordant  calls  to  STS  (up  to  11  consecu?ve  Cs)    Some  dele?ons  outside  C  tracts  observed  together  with  (concordant)  subs?tu?ons  (alignment  algorithm)    Sokware  constantly  improving;  TMAP  aligner  shows  generally  good  performance,  paired  read  aligners  seem  to  perform  beser  when  indels  are  present  in  homopolymeric  regions  with  respect  to  the  rCRS  

Summary  and  outlook  

Acknowledgements

FP7-­‐SEC-­‐2011-­‐285487  

Transla?onal  Research  project  L397    “EMPOP–an  innova?ve  human  mtDNA  database”    

2011-­‐MU-­‐MU-­‐K402  Maximizing  mtDNA  Tes?ng  Poten?al  with  the  Genera?on    of  High-­‐Quality  mtGenome  Reference  Data  

Robert  Lagacé  Sharon  Wooson  Reina  Langit  

Chris?na  Strobl  Gabriela  Huber  Bexna  Zimmermann  Liane  Fendt  

Samples  Sibylle  Marcial  Gomes,  Luis  Souto,  University  of  Aveiro,  Portugal  Rhena  Delport,  University  of  Pretoria,  South  Africa    

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