technology transition workshop| thomas hall, ph.d
TRANSCRIPT
TechnologyTransition Workshop
Overview of the Ibis™ mtDNA Tiling Assay
Technology Transition Workshop| Thomas Hall, Ph.D.
TechnologyTransition Workshop
Outline
• Ibis™ mtDNA assay format
• Base composition analysis of mtDNA samples− Data processing and analysis
− Blinded samples
• Information content relative to sequencing
• Heteroplasmy detection
• Sensitivity
• Reproducibility
Forensic SNP Analysis 2Overview of the Ibis mtDNA Tiling Assay
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• 24 primer pairs cover amplified coordinates HV1 15924..16428 and HV2 31..576• Target most highly‐conserved positions on 3′ ends of primers• Grouped into eight triplexed sets (colored grouping) by maximum spatial
separation and suitable mass separation of products
mtDNA Tiling Assay Format
(middle of mtDNA hidden)
HV1 15893..16451 (15924..16428 amplified)
HV2 5..603 (31..576 amplified)
Forensic SNP Analysis 3Overview of the Ibis mtDNA Tiling Assay
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mtDNA Tiling Assay Format
• Primers grouped to maximize target site separation
• PCR reactions performed with short extension cycle (five seconds)
• Product masses resolve from each other in triplex groupings
• Product sizes range from 85 to 140 bp; all but three are <130 bp
• Relative primer pair concentrations in triplexes have been adjusted to favor simultaneous amplification of all products
HV1 HV2
Forensic SNP Analysis 4Overview of the Ibis mtDNA Tiling Assay
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Base Composition Analysis
1. PCR
2. Desalting
3. ESI‐TOF mass spectrometry
4. Raw spectrum processing / deconvolution
5. Base composition assignment / profile development
Forensic SNP Analysis 5Overview of the Ibis mtDNA Tiling Assay
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HV1 HV2
PCR: Three Primer Pairs Per Reaction
Forensic SNP Analysis 6Overview of the Ibis mtDNA Tiling Assay
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Desalting of PCR ReactionsMagnetic bead anion exchange
Forensic SNP Analysis 7Overview of the Ibis mtDNA Tiling Assay
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ESI‐TOF Mass Spectromery• Three primer pairs per reaction• Complex raw spectrum
Forensic SNP Analysis 8Overview of the Ibis mtDNA Tiling Assay
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ESI‐TOF Mass Spectromery• Three primer pairs per reaction• Complex raw spectrum
• Multiple peaks for each mass• 3 (or more) ds products per
spectrum• 6 DNA strands per spectrum
Forensic SNP Analysis 9Overview of the Ibis mtDNA Tiling Assay
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ESI‐TOF Mass Spectromery• Three primer pairs per reaction• Complex raw spectrum
• Multiple peaks for each mass• 3 (or more) ds products per
spectrum• 6 DNA strands per spectrum
Noise baseline
Forensic SNP Analysis 10Overview of the Ibis mtDNA Tiling Assay
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ESI‐TOF Mass Spectromery• Three primer pairs per reaction• Complex raw spectrum
• Multiple peaks for each mass• 3 (or more) ds products per
spectrum• 6 DNA strands per spectrum
Baseline corrected spectrum used for deconvolution
Forensic SNP Analysis 11Overview of the Ibis mtDNA Tiling Assay
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Raw Spectrum ProcessingInternal mass standards bracket the spectrum for accurate calibration of the measurements before deconvolution
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 12
Internal mass
standard
Internal mass
standard
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Internal mass
standard
Internal mass
standard
• Peaks for each mass correlated by spacing through spectrum
• Internal mass standards ensure correlation of the correct peaks
Spectral Deconvolution
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 13
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Deconvolution to Masses
32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Signal intensity
Deconvolution results in one final measurement per molecular species
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 14
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DS DNA Strand AssociationForward and reverse strands of a double stranded
DNA can be associated by mass
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 15
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Signal intensity
32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da
TechnologyTransition Workshop
DS DNA Strand AssociationForward and reverse strands of a double stranded
DNA can be associated by mass
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 16
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,000
1000
2000
3000
4000
5000
6000
Mass (Da)
Signal intensity
32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da
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Base Composition Assignment
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,000
1000
2000
3000
4000
5000
6000
Mass (Da)
Signal intensity
32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 17
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30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Sig
nal i
nten
sity
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
Base Composition Assignment
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 18
TechnologyTransition Workshop
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Sig
nal i
nten
sity
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
Final Product Assignment
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 19
TechnologyTransition Workshop
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Sig
nal i
nten
sity
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
15893..16012[A47 G18 C25 T30]
Final Product Assignment
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 20
TechnologyTransition Workshop
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Sig
nal i
nten
sity
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
15893..16012[A47 G18 C25 T30]
Final Product Assignment
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 21
TechnologyTransition Workshop
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Sig
nal i
nten
sity
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
15893..16012[A47 G18 C25 T30]
16231..16338[A40 G9 C40 T19]
Final Product Assignment
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 22
TechnologyTransition Workshop
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Sig
nal i
nten
sity
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
15893..16012[A47 G18 C25 T30]
16231..16338[A40 G9 C40 T19]
Final Product Assignment
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 23
TechnologyTransition Workshop
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
6000
Mass (Da)
Sig
nal i
nten
sity
3285
5.1
3414
3.8
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
15893..16012[A47 G18 C25 T30]
16231..16338[A40 G9 C40 T19]
154..290[A49 G17 C31 T40]
Final Product Assignment
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 24
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Eight Spectra Per Sample
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 25
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Coverage Map Development
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 26
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2901: 15893..16012: A47 G18 C25 T302925: 15937..16041: A35 G14 C24 T322899: 15985..16073: A26 G15 C21 T272898: 16025..16119: A26 G17 C26 T262897: 16055..16155: A31 G13 C30 T272896: 16102..16224: A45 G13 C42 T232895: 16130..16224: A36 G7 C33 T192893: 16154..16268: A44 G7 C46 T182892: 16231..16338: A40 G9 C40 T192891: 16256..16366: A37 G9 C41 T242890: 16318..16402: A20 G14 C30 T212889: 16357..16451: A21 G17 C36 T212902: 5..97: A19 G24 C24 T262903: 20..139: A24 G34 C29 T332904: 83..187: A23 G21 C29 T322905: 113..245: A39 G18 C28 T482906: 154..290: A49 G17 C31 T402908: 204..330: A42 G16 C35 T322908: 204..330: A42 G16 C36 T322908: 204..330: A42 G16 C37 T322908: 204..330: A42 G16 C38 T322907: 239..363: A43 G11 C46 T232907: 239..363: A43 G11 C47 T232907: 239..363: A43 G11 C48 T232907: 239..363: A43 G11 C49 T232923: 262..390: A47 G10 C50 T202923: 262..390: A47 G10 C51 T202923: 262..390: A47 G10 C52 T202923: 262..390: A47 G10 C53 T202910: 331..425: A33 G9 C27 T262916: 367..463: A27 G8 C32 T302912: 409..521: A32 G7 C48 T262913: 464..603: A44 G10 C63 T23
Primer pair
rCRS coordinates
Base composition
Base composition
profile
Base Composition Profile
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 27
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490.41 1161.32 2117
m/z
0.00
37238
74476
490.43 1161.38 2117.12
m/z
0.00
56448.5
112897
490.44 1161.36 2117.06
m/z
0.00
26682
53364
490.44 1161.35 2117.04
m/z
0.00
40871
81742
490.43 1161.36 2117.09
m/z
0.00
39553
79106
490.43 1161.35 2117.06
m/z
0.00
31409.5
62819
490.43 1161.38 2117.12
m/z
0.00
46690
93380
490.41 1161.33 2117.04
0.00
39737.5
79475
490.42 1161.32 2117.01
m/z
0.00
33414.5
66829
490.43 1161.38 2117.12
m/z
0.00
52118
104236
490.44 1161.37 2117.1
m/z
0.00
29462
58924
490.43 1161.36 2117.08
m/z
0.00
50163
100326
490.43 1161.35 2117.06
m/z
0.00
34834.5
69669
490.43 1161.35 2117.06
m/z
0.00
34563.5
69127
490.43 1161.38 2117.12
m/z
0.00
44371
88742
490.44 1161.35 2117.04
0.00
35451
70902
490.43 1161.34 2117.03
m/z
0.00
33662.5
67325
490.43 1161.38 2117.12
m/z
0.00
47851.5
95703
490.43 1161.36 2117.08
m/z
0.00
26277
52554
490.44 1161.36 2117.06
m/z
0.00
45849
91698
490.42 1161.35 2117.07
m/z
0.00
34843.5
69687
490.44 1161.36 2117.08
m/z
0.00
31727
63454
490.43 1161.38 2117.12
m/z
0.00
34398
68796
490.43 1161.36 2117.08
0.00
40743.5
81487
490.43 1161.36 2117.08
m/z
0.00
27946
55892
490.43 1161.36 2117.09
m/z
0.00
52230
104460
490.43 1161.36 2117.08
m/z
0.00
24704.5
49409
490.44 1161.36 2117.08
m/z
0.00
55140.5
110281
490.42 1161.34 2117.05
m/z
0.00
34672
69344
490.44 1161.37 2117.1
m/z
0.00
32191.5
64383
490.43 1161.38 2117.12
m/z
0.00
28336.5
56673
490.42 1161.37 2117.13
0.00
39110
78220
490.43 1161.35 2117.06
m/z
0.00
34810.5
69621
490.44 1161.36 2117.08
m/z
0.00
53226
106452
490.42 1161.35 2117.07
m/z
0.00
29609.5
59219
490.44 1161.36 2117.06
m/z
0.00
47997
95994
490.43 1161.34 2117.03
m/z
0.00
36853
73706
490.43 1161.38 2117.12
m/z
0.00
32784.5
65569
490.43 1161.38 2117.12
m/z
0.00
27249.5
54499
490.43 1161.38 2117.12
0.00
43474.5
86949
490.44 1161.36 2117.08
m/z
0.00
24155
48310
490.42 1161.35 2117.07
m/z
0.00
56566.5
113133
490.41 1161.32 2117
m/z
0.00
30389
60778
490.43 1161.38 2117.12
m/z
0.00
50245.5
100491
490.41 1161.33 2117.04
m/z
0.00
32731.5
65463
490.43 1161.38 2117.12
m/z
0.00
34511
69022
490.43 1161.38 2117.12
m/z
0.00
28227
56454
490.43 1161.38 2117.12
0.00
39666
79332
490.43 1161.38 2117.12
m/z
0.00
42508.5
85017
490.42 1161.35 2117.07
m/z
0.00
51043
102086
490.41 1161.3 2116.96
m/z
0.00
30708.5
61417
490.43 1161.38 2117.12
m/z
0.00
42340.5
84681
490.41 1161.32 2117.02
m/z
0.00
39040.5
78081
490.43 1161.38 2117.12
m/z
0.00
32302.5
64605
490.44 1161.37 2117.1
m/z
0.00
39781.5
79563
490.44 1161.38 2117.11
0.00
39703
79406
490.43 1161.38 2117.12
m/z
0.00
22360
44720
490.43 1161.35 2117.06
m/z
0.00
52838
105676
490.42 1161.3 2116.95
m/z
0.00
24292.5
48585
490.42 1161.37 2117.13
m/z
0.00
49748
99496
490.41 1161.32 2117.02
m/z
0.00
35475.5
70951
490.44 1161.37 2117.1
m/z
0.00
31191
62382
490.43 1161.35 2117.06
m/z
0.00
30753
61506
490.44 1161.38 2117.12
0.00
38970
77940
490.43 1161.38 2117.14
m/z
0.00
35577
71154
490.43 1161.36 2117.08
m/z
0.00
52965
105930
490.42 1161.31 2116.98
m/z
0.00
30265.5
60531
490.43 1161.38 2117.12
m/z
0.00
42444
84888
490.41 1161.32 2117.02
m/z
0.00
34013.5
68027
490.44 1161.35 2117.04
m/z
0.00
30064
60128
490.42 1161.33 2117.03
m/z
0.00
36791
73582
490.44 1161.39 2117.14
0.00
40028.5
80057
490.44 1161.38 2117.11
m/z
0.00
47655.5
95311
490.44 1161.36 2117.08
m/z
0.00
47604
95208
490.41 1161.32 2117
m/z
0.00
31183.5
62367
490.43 1161.38 2117.12
m/z
0.00
39698.5
79397
490.41 1161.32 2117.02
m/z
0.00
35670
71340
490.43 1161.37 2117.11
m/z
0.00
30094
60188
490.42 1161.32 2117.01
m/z
0.00
28836
57672
490.45 1161.39 2117.13
0.00
39422.5
78845
490.44 1161.38
m/z
0.00
37610.5
75221
490.43 1161.36
m/z
0.00
47323
94646
490.41 1161.32
m/z
0.00
27089
54178
490.43 1161.38
m/z
0.00
43809.5
87619
490.42 1161.33
m/z
0.00
38638
77276
490.44 1161.37
m/z
0.00
35201
70402
490.41 1161.32
m/z
0.00
30036.5
60073
490.44 1161.39
0.00
50655
101310
Multiple Assays Per Plate
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 28
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Multiple Mass Assignments
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 29
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Multiple Samples Per Plate
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 30
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2901: 15893..16012: A47 G18 C25 T302925: 15937..16041: A35 G14 C24 T322899: 15985..16073: A26 G15 C21 T272898: 16025..16119: A26 G17 C26 T262897: 16055..16155: A31 G13 C30 T272896: 16102..16224: A45 G13 C42 T232895: 16130..16224: A36 G7 C33 T192893: 16154..16268: A44 G7 C46 T182892: 16231..16338: A40 G9 C40 T192891: 16256..16366: A37 G9 C41 T242890: 16318..16402: A20 G14 C30 T212889: 16357..16451: A21 G17 C36 T212902: 5..97: A19 G24 C24 T262903: 20..139: A24 G34 C29 T332904: 83..187: A23 G21 C29 T322905: 113..245: A39 G18 C28 T482906: 154..290: A49 G17 C31 T402908: 204..330: A42 G16 C35 T322908: 204..330: A42 G16 C36 T322908: 204..330: A42 G16 C37 T322908: 204..330: A42 G16 C38 T322907: 239..363: A43 G11 C46 T232907: 239..363: A43 G11 C47 T232907: 239..363: A43 G11 C48 T232907: 239..363: A43 G11 C49 T232923: 262..390: A47 G10 C50 T202923: 262..390: A47 G10 C51 T202923: 262..390: A47 G10 C52 T202923: 262..390: A47 G10 C53 T202910: 331..425: A33 G9 C27 T262916: 367..463: A27 G8 C32 T302912: 409..521: A32 G7 C48 T262913: 464..603: A44 G10 C63 T23
2901: 15893..16012: A46 G19 C25 T302925: 15937..16041: A35 G14 C24 T322899: 15985..16073: A26 G15 C21 T272898: 16025..16119: A26 G17 C28 T242897: 16055..16155: A32 G12 C30 T272896: 16102..16224: A46 G12 C41 T242895: 16130..16224: A36 G7 C33 T192893: 16154..16268: A44 G7 C45 T192892: 16231..16338: A40 G9 C40 T192891: 16256..16366: A37 G9 C41 T242890: 16318..16402: A20 G14 C30 T212889: 16357..16451: A22 G16 C36 T212902: 5..97: A19 G24 C24 T262903: 20..139: A24 G34 C29 T332904: 83..187: A23 G21 C30 T312905: 113..245: A39 G18 C31 T452906: 154..290: A48 G18 C35 T362908: 204..330: A42 G16 C40 T302907: 239..363: A43 G11 C49 T232923: 262..390: A47 G10 C53 T202910: 331..425: A33 G9 C27 T262916: 367..463: A27 G8 C32 T302912: 409..521: A32 G7 C48 T262913: 464..603: A45 G10 C68 T232913: 464..603: A45 G10 C69 T23
2901: 15893..16012: A47 G18 C25 T302925: 15937..16041: A35 G14 C24 T322899: 15985..16073: A26 G15 C21 T272898: 16025..16119: A26 G17 C27 T252897: 16055..16155: A32 G12 C29 T282897: 16055..16155: A31 G13 C29 T282896: 16102..16224: A46 G12 C42 T232896: 16102..16224: A45 G13 C42 T232895: 16130..16224: A36 G7 C33 T192893: 16154..16268: A44 G7 C46 T182892: 16231..16338: A39 G10 C40 T192891: 16256..16366: A36 G10 C42 T232890: 16318..16402: A20 G14 C30 T212889: 16357..16451: A21 G17 C36 T212902: 5..97: A20 G23 C24 T262903: 20..139: A25 G33 C29 T332904: 83..187: A23 G21 C29 T322905: 113..245: A39 G18 C29 T472906: 154..290: A48 G18 C32 T392908: 204..330: A42 G16 C39 T322907: 239..363: A43 G11 C50 T232907: 239..363: A43 G11 C51 T232923: 262..390: A47 G10 C54 T202923: 262..390: A47 G10 C55 T202910: 331..425: A33 G9 C27 T262916: 367..463: A27 G8 C32 T302912: 409..521: A32 G7 C48 T262913: 464..603: A44 G10 C63 T23
Multiple Profiles Per Plate
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 31
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Blinded Samples• AFDIL
− 25 dried blood spots on filter paper
• FBI− 25 buccal swabs− Samples chosen to test system because each had
mismatches in at least one primer pair• NIST
− 95 population reference samples• All reactions were run on pre‐fabricated, frozen PCR
plates (just add template)• Batch of plates had been through a QC test prior to use• Each assay had a least one positive control• Each plate had at least one negative control
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 32
TechnologyTransition Workshop
Blinded Samples• All samples gave complete profiles• Three FBI samples were not pure single‐contributor templates− Two FBI samples were mixed
• Both were consistent with one sample in primary set mixed with another sample not in the set
• FBI confirmed correct identification of mixtures• Approximate ratios of contributing templates were quantified
− One FBI sample was a blank swab• Several heteroplasmies, both C‐length and non‐C‐length were noted in all sample sets − Consistent with sequence profile data
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 33
TechnologyTransition Workshop
Tiling Compared to Sequencing• 1266 unique tiling region sequences were selected from GenBank genomes− Each sequence differed from all others by at least one base
− C‐stretch length differences ignored• Sequences converted to tiling base compositions• Cross‐compared for minimum differences using mtDNA search algorithm− Ignores C‐stretch length differences− Corrects for primer pair overlaps
• 94.2% of unique tiling region sequences were uniquely discriminated by tiling assay
Forensic SNP Analysis Overview of the Ibis mtDNA Tiling Assay 34
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Tiling Compared to Sequencing
CRSTiling assay amplifies 15924‐16428 and 31‐576
Minimum HVI +HVII sequences 16024‐16365 and 73‐340
16024 16365 73 340
For the same set of 1266 unique sequences spanning mtDNA tiling coordinates:
• 94.2% can be differentiated with the tiling assay• 90.2% can be differentiated by sequencing
HVI 16024‐16365 and HV2 73‐340
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Length Heteroplasmy Detection
Good sequence up to C stretch, then multiple overlapping reads
MS detects multiple products while sequencing requires a pure sample for
maximum information content
287.
9
C
338.
7
G*
289.
9
C
339.
5
G*
289.
2
C
339.
3
G*
3724
1.0
3845
7.9
A44 G13 C42 T23
3752
8.9
3879
6.6
A44 G13 C43 T23
3781
8.8 3913
6.1
A44 G13 C44 T23
3810
8.0
3947
5.4
A44 G13 C45 T23
Happened to be same sample given as a blinded sample by Bruce Budowle in 2003.This was suggested by our assay in the FBI validation, then confirmed.
*
Example = USA.FBI.000009, region 16102..16224 *
Opposite read
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Length Heteroplasmy Example2901: 15893..16012: A47 G18 C25 T302925: 15937..16041: A35 G14 C24 T322899: 15985..16073: A25 G16 C21 T272898: 16025..16119: A25 G18 C27 T252897: 16055..16155: A31 G12 C30 T282896: 16102..16224: A44 G12 C42 T242896: 16102..16224: A44 G12 C43 T242896: 16102..16224: A44 G12 C44 T242896: 16102..16224: A44 G12 C45 T242895: 16130..16224: A35 G7 C33 T192895: 16130..16224: A35 G7 C34 T192895: 16130..16224: A35 G7 C35 T192895: 16130..16224: A35 G7 C36 T192893: 16154..16268: A43 G7 C47 T172893: 16154..16268: A43 G7 C48 T172893: 16154..16268: A43 G7 C49 T172893: 16154..16268: A43 G7 C50 T172892: 16231..16338: A40 G9 C39 T202891: 16256..16366: A37 G9 C41 T242890: 16318..16402: A20 G14 C31 T202889: 16357..16451: A21 G17 C36 T212902: 5..97: A19 G24 C24 T262903: 20..139: A24 G34 C29 T332904: 83..187: A23 G21 C30 T312905: 113..245: A39 G18 C30 T462905: 113..245: A39 G18 C29 T472906: 154..290: A48 G18 C32 T392906: 154..290: A48 G18 C31 T402908: 204..330: A42 G16 C39 T322908: 204..330: A42 G16 C40 T322907: 239..363: A43 G11 C49 T242907: 239..363: A43 G11 C50 T242923: 262..390: A47 G10 C53 T212923: 262..390: A47 G10 C54 T212910: 331..425: A33 G9 C27 T262916: 367..463: A27 G8 C32 T302912: 409..521: A32 G7 C48 T262913: 464..603: A43 G11 C63 T23
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A45 G13 C41 T24
16102..16224: A45 G13 C40 T24 N
16130..16224: A36 G7 C32 T19 N
16102..16224: A45 G13 C41 T2416102..16224: A45 G13 C40 T2516130..16224: A36 G7 C33 T1916130..16224: A36 G7 C32 T20
Observed profile Calculated from truth key
C/T heteroplasmy
C/T heteroplasmyAF‐4: 16024‐16365
C 16176 N
T 16362 C
37582.4 Da
PP 2896: 16102..16224 38710.7 Da
A45 G13 C41 T24
A45 G13 C40 T25
37569.3 Da
38736.4 Da
From sequence profile
From blinded sample AF‐4
37,600 37,800 38,000 38,200 38,400 38,600 38,8000
500
1000
1500
2000
Mass (Da)
Intensity
SNP Heteroplasmy Detection
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SNP Heteroplasmy Detection• SNP heteroplasmies observed in blinded samples plus 21 in‐house
samples used for comparison to sequence data
• 21 in‐house samples had full control‐region sequence data
• Other samples had sequence covering 12 to 22 of 24 primer pairs
• All profiles were consistent with sequence data
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Sensitivity• Analysis of five templates in dilution‐to‐extinction• Sensitivity ranged from 0.1 pg to 6.3 pg template / primer pair*• All templates had full profile at 6.3 pg or below per reaction• Sensitivity criteria for standard QC plate set at 25 pg / reaction
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* Based upon total nuclear DNA quantification with Quantifiler®
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Reproducibility• 3,331 trials of one positive control
template run over the course of 27 months
• 500 pg template / reaction
• Automated data analysis
• 79,944 expected ds assignments
• 3,298 (99%) full, correct profiles
• 33 (1%) samples missing primer pair(s)
• 85 total missed assignments (0.11%)
• 113 artifact assignments (0.14%)− All but 10 (0.013%) correctable
with manual QC
• 21 mis‐assignments (0.026%)− All but 12 (0.015%) correctable
with manual QC
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Ave error magnitude was 10.12 + 8.04 ppm
Distribution of mass measurement deviations for
159,688 DNA strand assignments
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Summary• 1051 nucleotide positions covered by 24 primer pairs• Accurate mass measurements and biochemical strategy
allow mitochondrial base composition profiles to be developed
• Discrimination power is about 94% that of sequencing same region
• Discrimination power over tiling region can be greater than sequencing over minimum HV1 and HV2 ranges
• Base composition profiles can be compared to each other and to sequence profiles
• Databases can be searched and subjected to same type of statistics as a sequence database
• Mass spectrometry can resolve heteroplasmy (mixtures)
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Funded in part by NIJ contract2006‐DN‐BX‐K011
Questions?
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Contact Information
Thomas Hall
Ibis Biosciences division of Abbott Molecular
760‐603‐2375
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Note: All images and charts courtesy of Tom Hall, Ph.D.