Technology Transition Workshop

Overview of the Ibis mtDNA Tiling Assay

Tom Hall, Ph.D.Ibis Biosciences, Inc.

Technology Transition WorkshopOutline

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

Technology Transition Workshop

mtDNA Tiling Assay Format 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 8 triplexed sets (colored grouping) by maximum

spatial separation and suitable mass separation of products

(middle of mtDNA hidden)

HV1 15893..16451 (15924..16428 amplified)

HV2 5..603 (31..576 amplified)

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

HV1 HV2 Primers grouped to maximize target site separation PCR reactions performed with short extension cycle (5 seconds)

Product sizes range from 85 to 140 bp. All but 3 are <130 bp Relative primer pair concentrations in triplexes have been

adjusted to favor simultaneous amplification of all products

Product masses resolve from each other in triplex groupings

mtDNA Tiling Assay Format

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

Base Composition Analysis1. PCR

2. Desalting

3. ESI-TOF mass spectrometry

4. Raw spectrum processing / deconvolution

5. Base composition assignment / profile

development

Technology Transition Workshop

HV1 HV2

PCR: 3 Primer Pairs Per Reaction

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition WorkshopDesalting of PCR Reactions

AddDNA to beads

Bind DNA

MagneticPull-down

Remove buffer

Washes

Elute DNA

Magnetically pull out beads

Remove DNA / Mass Spec

Remove salt from

DNA bound to

beads

Remove clean

DNA for analysis

Magnetic bead anion exchange

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition WorkshopESI-TOF Mass Spectromery

Three primer pairs per reactionComplex raw spectrum

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

• Multiple peaks for each mass

• 3 (or more) ds products per spectrum

• 6 DNA strands per spectrum

Noise baseline

Baseline corrected spectrum used for deconvolution

ESI TOF Mass SpectromeryThree primer pairs per reactionComplex raw spectrum

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

Internal mass

standard

Internal mass

standard

Internal mass standards bracket the spectrum for accurate calibration of the measurements before deconvolution

Raw Spectrum Processing

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

Deconvolution results in one final measurement per molecular species

32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da

Deconvolution to Masses

30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000

1000

2000

3000

4000

5000

6000

Mass (Da)

Sign

al in

tens

ity

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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)

Sign

al in

tens

ityForward and reverse strands of a double stranded DNA can be associated by mass

32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da

DS DNA Strand Association

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,000

1000

2000

3000

4000

5000

6000

Mass (Da)

Sign

al in

tens

ityForward and reverse strands of a double stranded DNA can be associated by mass

32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da

DS DNA Strand Association

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

44,000

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)

Sign

al in

tens

ity

32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da

Base Composition Assignment

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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]

Base Composition Assignment

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition 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

IMAGE COURTESYOF TOM HALL, PH.D.

Technology Transition WorkshopEight Spectra Per Sample

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition WorkshopCoverage Map Development

IMAGES COURTESYOF TOM HALL, PH.D.

Technology Transition Workshop

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

Base Composition Profile

Primer pairrCRS coordinatesBase composition

Base composition

profile

IMAGES COURTESYOF TOM HALL, PH.D.

Technology Transition Workshop

Multiple Assays Per Plate

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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

Multiple Mass Assignments1 (A01)

2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

2776.5

5553

13 (B01)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

3153

6306

25 (C01)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2443

4886

37 (D01)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

2083

4166

49 (E01)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

2513

5026

61 (F01)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

1983

3966

73 (G01)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2560

5120

85 (H01)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

2268.5

4537

2 (A02)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

2433.5

4867

14 (B02)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

2581.5

5163

26 (C02)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2475.5

4951

38 (D02)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

2113

4226

50 (E02)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

2033

4066

62 (F02)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

1829

3658

74 (G02)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2456

4912

86 (H02)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

1929

3858

3 (A03)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

2433

4866

15 (B03)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

2485

4970

27 (C03)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2198.5

4397

39 (D03)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

2020.5

4041

51 (E03)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

2209.5

4419

63 (F03)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

1762.5

3525

75 (G03)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2511

5022

87 (H03)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

2101

4202

4 (A04)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

1250.5

2501

16 (B04)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

1538

3076

28 (C04)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

1239

2478

40 (D04)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

1282

2564

52 (E04)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

1253

2506

64 (F04)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

638

1276

76 (G04)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

1248

2496

88 (H04)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

1099

2198

5 (A05)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

2610

5220

17 (B05)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

2975.5

5951

29 (C05)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2595

5190

41 (D05)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

2296

4592

53 (E05)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

2374

4748

65 (F05)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

2143.5

4287

77 (G05)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2109

4218

89 (H05)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

2384

4768

6 (A06)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

2108

4216

18 (B06)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

2385.5

4771

30 (C06)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2042.5

4085

42 (D06)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

1255

2510

54 (E06)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

1683

3366

66 (F06)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

1665

3330

78 (G06)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2064

4128

90 (H06)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

1768

3536

7 (A07)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

3185

6370

19 (B07)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

3194

6388

31 (C07)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2917.5

5835

43 (D07)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

2419

4838

55 (E07)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

2660

5320

67 (F07)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

2407

4814

79 (G07)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2431.5

4863

91 (H07)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

2555

5110

8 (A08)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

1702.5

3405

20 (B08)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

1990

3980

32 (C08)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

1527

3054

44 (D08)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

1644

3288

56 (E08)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

1555.5

3111

68 (F08)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

1430

2860

80 (G08)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

1871.5

3743

92 (H08)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

1502.5

3005

9 (A09)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

2729.5

5459

21 (B09)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

2802.5

5605

33 (C09)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2502

5004

45 (D09)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

1451

2902

57 (E09)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

2325

4650

69 (F09)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

1804.5

3609

81 (G09)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2302.5

4605

93 (H09)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

2008

4016

10 (A10)2906 + 2901 + 2892

23000 36999.75 50999.51

Mass (Da)

0.00

3169

6338

22 (B10)2925 + 2891 + 2908

23000 36999.75 50999.51

Mass (Da)

0.00

3076.5

6153

34 (C10)2899 + 2890 + 2907

23000 36999.75 50999.51

Mass (Da)

0.00

2867.5

5735

46 (D10)2898 + 2889 + 2923

23000 36999.75 50999.51

Mass (Da)

0.00

2266.5

4533

58 (E10)2893 + 2910 + 2902

23000 36999.75 50999.51

Mass (Da)

0.00

2621.5

5243

70 (F10)2897 + 2903 + 2916

23000 36999.75 50999.51

Mass (Da)

0.00

2073

4146

82 (G10)2896 + 2913 + 2904

23000 36999.75 50999.51

Mass (Da)

0.00

2634.5

5269

94 (H10)2905 + 2895 + 2912

23000 36999.75 50999.51

0.00

2332.5

4665

11 (A11)2906 + 2901 + 2892

23000 36999.75 5

Mass (Da)

0.00

2881.5

5763

23 (B11)2925 + 2891 + 2908

23000 36999.75 5

Mass (Da)

0.00

2826.5

5653

35 (C11)2899 + 2890 + 2907

23000 36999.75 5

Mass (Da)

0.00

2566.5

5133

47 (D11)2898 + 2889 + 2923

23000 36999.75 5

Mass (Da)

0.00

2177

4354

59 (E11)2893 + 2910 + 2902

23000 36999.75 5

Mass (Da)

0.00

2488

4976

71 (F11)2897 + 2903 + 2916

23000 36999.75 5

Mass (Da)

0.00

2171

4342

83 (G11)2896 + 2913 + 2904

23000 36999.75 5

Mass (Da)

0.00

2559

5118

95 (H11)2905 + 2895 + 2912

23000 36999.75 5

0.00

2429.5

4859

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

Multiple Samples Per Plate

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

Multiple Profiles Per Plate

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

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

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 has a least one positive control Each plate has at least one negative control

Blinded Samples

Technology Transition Workshop

All samples gave complete profiles 3 FBI samples were not pure single-contributor

templates 2 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 1 FBI sample was a blank swab

Several heteroplasmies, both C-length and non-C-length were noted in all sample sets and were consistent with sequence profile data

Blinded Samples

Technology Transition Workshop

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

Tiling Compared to Sequencing

Technology Transition Workshop

CRS Tiling assay amplifies 15924-16428 and 31-576Minimum HVI +HVII sequences 16024-16365 and 73-34016024 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

Tiling Compared to SequencingIMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

37,000 37,500 38,000 38,500 39,000 39,500 40,000

200

400

600

800

1000

1200

1400

Mass (Da)

Sign

al in

tens

ity

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 38

796.

6A44 G13 C43 T23

3781

8.8 39

136.

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

Length Heteroplasmy Detection

IMAGES COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

2901: 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

Length Heteroplasmy ExampleIMAGES COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

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: 16024-16365C 16176 NT 16362 C

3758

2.4

Da

PP 2896: 16102..16224 3871

0.7

Da

A45 G13 C41 T24A45 G13 C40 T25

3756

9.3

Da

3873

6.4

Da

From sequence profile

SNP Heteroplasmy Detection

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)

Inte

nsity

IMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition Workshop

Contributor SampleHetero-plasmy

Replicates in

observed region

Var. 1

Percentage of variant 1

Var. 2

Percentage of variant 2

Primer pairs

Amplified overlap

Sequence data

CS0021 C<->T 4 T 14.5 ± 0.6 C 85.5 ± 0.6 2898, 2897 16,078 - 16,098 16,093 YCS0038 C<->T 1 T 25.4 C 74.6 2891 16,283 - 16,344 16,325 YCS0040 C<->T 2 T 35.7 ± 0.3 C 64.3 ± 0.3 2898, 2897 16,078 - 16,098 16,093 Y

AF-2 C<->T 4 T 30.0 ± 2.2 C 70.0 ± 2.2 2892, 2891 16,283 - 16,305 16,293 NAF-4 C<->T 4 T 48.6 ± 2.2 C 51.4 ± 2.2 2896, 2895 16,157 - 16,201 16,176 NAF-15 A<->G 4 A 21.1 ± 2.0 G 78.9 ± 2.0 2897, 2896 16,124 - 16,129 NDAF-19 A<->G 4 G 44.8 ± 1.1 A 55.2 ± 1.1 2899, 2898 16,048 - 16,051 16,051 NFBI-37 C<->T 4 T 22.9 ± 0.8 C 77.1 ± 0.8 2892, 2891 16,283 - 16,305 16,298 YFBI-47 C<->T 4 T 17.3 ± 0.6 C 82.7 ± 0.6 2898, 2897 16,078 - 16,098 16,093 YFBI-51 C<->T 2 T 45.9 +- 4.2 C 54.1 +- 4.2 2902, 2903 41 - 76 64 YFBI-66 C<->T 4 C 48.5 ± 1.1 T 51.5 ± 1.1 2905, 2906 178 - 217 195 YFBI-72 C<->T 4 T 39.8 ± 4.1 C 60.2 ± 4.1 2905, 2906 178 - 217 217 Y

NIST-JT51499 C<->T 4 T 41.4 ± 0.4 C 58.6 ± 0.4 2905, 2906 178 - 217 198 YNIST-JT52076 C<->T 2 C 34.9 ± 0.1 T 65.1 ± 0.1 2892 16,254 - 16,305 16,260 YNIST-PT84224 C<->T 4 T 48.3 ± 0.8 C 51.7 ± 0.8 2898, 2897 16,078 - 16,098 NDNIST-PT84231 C<->T 4 T 19.1 ± 0.5 C 80.9 ± 0.5 2905, 2906 178 - 217 204 Y

In-house

AFDIL

FBI

NIST

SNP Heteroplasmy DetectionSNP 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 dataOther samples had sequence covering 12 to 22 of 24 primer pairsAll profiles were consistent with sequence data

Technology Transition WorkshopSensitivity

Analysis of 5 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

288928902891289228932895289628972898289929252901290229032904290529062907290829232910291629122913

Prim

er P

air

100 pg 25 pg 6.3 pg 1.6 pg 0.1 pg0.4 pg

min

max

50 pg 12.5 pg 3.2 pg 0.8 pg 0.2 pg

* Based upon total nuclear DNA quantification with Quantifiler.

GRAPH COURTESY OF TOM HALL, PH.D.

Technology Transition WorkshopReproducibility

0

10000

20000

30000

40000

50000

-85 to -75-75 to -65-65 to -55-55 to -45-45 to -35-35 to -25-25 to -15-15 to -5-5 to 55 to 1515 to 2525 to 3535 to 4545 to 5555 to 6565 to 7575 to 85

3,331 trials of one positive control template run over the course of 27 months

500 pg template / reaction

79,944 expected ds assignments Automated data analysis

Distribution of mass measurement deviations for 159,688 DNA strand

assignmentsAve error magnitudewas 10.12 + 8.04 ppm

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 misassignments (0.026%) All but 12 (0.015%) correctable

with manual QCIMAGE COURTESY OF TOM HALL, PH.D.

Technology Transition WorkshopSummary

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 ~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)

Technology Transition Workshop

Contact Information:

Tom Hall, Ph.D.Ibis Biosciences, Inc.thall@ibisbio.com