next generation mtgenome sequencing for forensic purposes using the ion torrent pgm
DESCRIPTION
TRANSCRIPT
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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
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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
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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)
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Sanger-‐type sequencing of mtGenomes
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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
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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)
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Inspec*on of PGM data – Sequencher Tablet
rCRS
Coverage
Sequences
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Inspec*on of PGM data – Sequencher Tablet
diff-‐coded (rCRS) forward -‐ reverse nucleo>des
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Inspec*on of PGM data – IGV
Total Count: 667 A: 0 C: 5 (1%) G: 0 T: 662 (99%) N: 0 INS: 5
rCRS
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Analysis tools – Ion Torrent Server mito plug-‐in
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Analysis tools – Ion Torrent Server mito plug-‐in
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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
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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)
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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
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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
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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
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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
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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
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Acknowledgements
Euroforgen FP7-‐SEC-‐2011-‐285487
Transla*onal Research project L397 “EMPOP–an innova*ve human mtDNA database”
2010-‐91361-‐DC-‐DN