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

 Strobl  C1,  Huber  G1,  Lagacé  R2,  Langit  R2,  Woo9on  S2,  Hennessy  L2,  Parson  W1  1Ins>tute  for  Legal  Medicine  Innsbruck,  Innsbruck,  Austria  

2Life  Technologies,  Foster  City,  United  States  

DNA  in  Forensics  2012,  Sep  08  2012    5th  Interna>onal  EMPOP  Mee>ng  -­‐  8th  Interna>onal  Forensic  Y-­‐User  Workshop    

Why  mtGenomes?  

advoca>ng  for  research  on  NGS  in  the  forensic  field    extending  QC  mechanisms  established  for  the  control  region  

2010-­‐91361-­‐DC-­‐DN  Maximizing  mtDNA  Tes>ng  Poten>al  with  the  Genera>on    of  High-­‐Quality  mtGenome  Reference  Data  

Why  PGM?  

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)  

Sanger-­‐type  sequencing  of  mtGenomes  

PGM  NGS  of  mtGenomes

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

PCR   e-­‐shearing  (130-­‐140  bp)  

100  bp  chemistry  316  chips  

Analysis  tools

Consensus  sequence  coverage  dependent  on  alignment  soKware    Torrent  Browser  (Ion  Torrent)  

 Sofware  suite  that  creates  different  output  formats  of  the  sequences    e.g.  Variant  Caller  (V2;  V3  expected  in  Sep  12)    uses  TMAP  Smith-­‐Waterman  alignment  op>miza>on  (Li  and  Homer,  2010)  

     

Sequencher  Tablet  (GeneCodes)    Viewer  for  NGS  integrated  in  Sequencher    Uses  GSNAP  for  alignment  (Wu  and  Nacu,  2010)  

   Integra>ve  Genomics  Viewer  (IGV)  

 Freeware  to  visualize  alignment  files  (Robinson  et  al,  2011)      

Inspec*on  of  PGM  data  –  Sequencher  Tablet

rCRS    

Coverage    

Sequences    

Inspec*on  of  PGM  data  –  Sequencher  Tablet

diff-­‐coded  (rCRS)     forward  -­‐  reverse     nucleo>des    

Inspec*on  of  PGM  data  –  IGV

Total Count: 667 A: 0 C: 5 (1%) G: 0 T: 662 (99%) N: 0 INS: 5

rCRS    

Analysis  tools  –  Ion  Torrent  Server  mito  plug-­‐in

Analysis  tools  –  Ion  Torrent  Server  mito  plug-­‐in

Comparison  of  haplotypes

Sanger PGM Position rCRS Sequencher Seq-T IT VC IT cons 73 A G G G G 152 T C C C C 263 A G G G G 295 C A A A A 309,1 : C n.d. n.d. n.d. 315,1 : C n.d. n.d. n.d. 497 C T T T T 750 A G G G G 1189 T C C C C 1438 A G G G G 1811 A G G G G 2706 A G G G G 3084 A G G G G 3480 A G G G G 4769 A G G G G 7028 C T T T T 8860 A G G G G 9055 G A A A A 9422 A G G G G 9698 T C C C C 10398 A G G G G 10550 A G G G G 11299 T C C C C 11467 A G G G G 11719 G A A A A 12308 A G G G G 12372 G A A A A 14167 C T T T T 14766 C T T T T 14798 T C C C C 15326 A G G G G 16224 T C C C C 16311 T C C C C 16519 T C C C C

Full  accordance  outside  HVS-­‐2  tract  

Haplogroup  K1a  

8C 6C T 6-8C 5C T

Comparison  of  haplotypes

Sanger PGM

Position rCRS Sequencher Seq-T IT VC IT cons 73 A G G G G

146 T C C C C

189 A G G G G

195 T C C C C

199 T C C C C

247 G A A A A

315,1 : C n.d. n.d. n.d.

498 C : n.d. n.d. n.d.

523 A : n.d. : :

524 C : n.d. : :

719 G A A A A

750 A G G G G

769 G A A A A

825 T A A A A

1018 G A A A A

1048 C T T T T

1692 A C C C C

2484,1 : C n.d. C C

2758 G A A A A

2885 T C C C C

3438 G A A A A

3516 C A A A A

3594 C T T T T

3618 T C C C C

3756 A G G G G

4104 A G G G G

4232 T C C C C

4312 C T T T T

4562 A C C C C

4769 A G G G G

5442 T C C C C

6185 T C C C C

6221 T C C C C

6266 A G G G G

6815 T C C C C

6998 C T T T T

7028 C T T T T

7146 A G G G G

7256 C T T T T

7521 G A A A A

Haplogroup  L0d1  1-­‐7521  

498del – coverage = 144)

Comparison  of  haplotypes

Sanger PGM

Position rCRS Sequencher Seq-T IT VC IT cons 73 A G G G G

146 T C C C C

189 A G G G G

195 T C C C C

199 T C C C C

247 G A A A A

315,1 : C n.d. n.d. n.d.

498 C : n.d. n.d. n.d.

523 A : n.d. : :

524 C : n.d. : :

719 G A A A A

750 A G G G G

769 G A A A A

825 T A A A A

1018 G A A A A

1048 C T T T T

1692 A C C C C

2484,1 : C n.d. C C

2758 G A A A A

2885 T C C C C

3438 G A A A A

3516 C A A A A

3594 C T T T T

3618 T C C C C

3756 A G G G G

4104 A G G G G

4232 T C C C C

4312 C T T T T

4562 A C C C C

4769 A G G G G

5442 T C C C C

6185 T C C C C

6221 T C C C C

6266 A G G G G

6815 T C C C C

6998 C T T T T

7028 C T T T T

7146 A G G G G

7256 C T T T T

7521 G A A A A

Haplogroup  L0d1  1-­‐7521  

498del – IGV

Comparison  of  haplotypes

Sanger PGM

Position rCRS Sequencher Seq-T IT VC IT cons 73 A G G G G

146 T C C C C

189 A G G G G

195 T C C C C

199 T C C C C

247 G A A A A

315,1 : C n.d. n.d. n.d.

498 C : n.d. n.d. n.d.

523 A : n.d. : :

524 C : n.d. : :

719 G A A A A

750 A G G G G

769 G A A A A

825 T A A A A

1018 G A A A A

1048 C T T T T

1692 A C C C C

2484,1 : C n.d. C C

2758 G A A A A

2885 T C C C C

3438 G A A A A

3516 C A A A A

3594 C T T T T

3618 T C C C C

3756 A G G G G

4104 A G G G G

4232 T C C C C

4312 C T T T T

4562 A C C C C

4769 A G G G G

5442 T C C C C

6185 T C C C C

6221 T C C C C

6266 A G G G G

6815 T C C C C

6998 C T T T T

7028 C T T T T

7146 A G G G G

7256 C T T T T

7521 G A A A A

Haplogroup  L0d1  1-­‐7521  

2484.1C – not captured with Seq T

Comparison  of  haplotypes

Haplogroup  L0d1  7522-­‐16569  

Sanger PGM Position rCRS Sequencher Seq-T IT VC IT cons 8113 C A A A A 8152 G A A A A 8251 G A A A A 8468 C T T T T 8655 C T T T T 8701 A G G G G 8860 A G G G G 9042 C T T T T 9347 A G G G G 9540 T C C C C 9755 G A A A A 10295 A G G G G 10589 G A A A A 10664 C T T n.d. n.d. 10688 G A A A A 10810 T C C C C 10873 T C C C C 10915 T C C C C 11719 G A A A A 11914 G A A A A 12007 G A A A A 12121 T C C C C 12705 C T T T T 12720 A G G G G 13105 A G G G G 13506 C T T T T 13650 C T T T T 13759 G A A A A 14766 C T T T T 15326 A G G G G 15466 G A A A A 15930 G A A A A 15941 T C C C C 16129 G A A A A 16179 C T T T T 16187 C T T T T 16189 T C C C C 16223 C T T T T 16230 A G G G G 16243 T C C C C 16311 T C C C C 16519 T C C C C

10664T – region close to primer B

Point  heteroplasmy

Haplogroup  H5r      

Sanger PGM Position rCRS Sequencher Seq-T IT VC IT cons 207 G A A A A 263 A G G G G 315,1 : C n.d. n.d. n.d. 456 C T T T T 750 A G G G G 1438 A G G G G 4769 A G G G G 8602 T - Y - - 8860 A G G G G 9966 G R R R R 10410 T C C C C 13725 C T T T T 15326 A G G G G 16304 T C C C C 16311 T C C C C

9966R

8602Y not present in Sanger or IGV

Preliminary  summary

Based  on  first  15  mtGenomes    General  high  accordance  between  sequencing  and  alignment  methods  under  standard  sofware  sekngs  –  inves>gate  customized  sekngs    Individual  differences  based  on  inser>on/dele>on  events  in  homopolymer  tracts  and/or  subs>tu>ons  close  to  primer  binding  sites  (alignment  method)    Homopolymeric  tracts  correctly  displayed  up  to  7  iden>cal  nucleo>des  (dependent  on  coverage)    Point  heteroplasmy  captured  in  all  cases  with  a  level  of  20%  (defined  threshold);  sequence  data  indicate  also  lower  levels  detectable  (dependent  on  coverage)      Coverage  important  for  quality  of  call,  determina>on  of  threshold  values  required    New  chemistry  upcoming  for  300  bp  sequencing  –  con>nue  evalua>on        

Acknowledgements

Euroforgen  FP7-­‐SEC-­‐2011-­‐285487  

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

2010-­‐91361-­‐DC-­‐DN