2016 degradation study - thermo fisher scientific - …...3 •unt: modified organic method •24...

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GlobalFiler™, PowerPlex Fusion™ 6C, PowerPlex Fusion 5C

2016 Degradation Study

2

• Goal

• Compare allele recovery and performance across a range of degraded samples

• Sample Types

• Bone Samples from University of North Texas (UNT)

• Cadaver Samples from Sam Houston State University (SHSU)

• Mock Samples: Buccal, Menstrual Blood and Tissue (Pulled Pubic Hair)

Weathered from Thermo Fisher Scientific (Known-K)

Introduction: Degradation Study

3

• UNT: Modified Organic Method

• 24 hour demineralization of bone powder (200 mg)

• 1% sarkosyl/EDTA solution (Sodium N-lauryl sarcosinate dissolved in 0.5M EDTA pH

8.0)

• Add fresh proteinase K (20 mg/ml) to the demin buffer before the overnight incubation

@ 56oC.

• Organic extraction

• Phenol Chloroform Isoamyl Alcohol (25:24:1)

• Purification and concentration

• Amicon Ultra-4 30K column

• Post extraction sample cleanup/inhibitor removal

• Qiagen QIAquick Kit

• Final volume of approximately 40-50 uL

Extraction Methods-UNT Bones

4

• SHSU: Soft Tissue Samples

• Digestion

• 20 mg of tissue

• Buffer ATL and Proteinase K (20mg/ml)

• Incubate at 56oC for one hour

• Purification

• Qiagen QIAamp™ FFPE Tissue Kit

Start with 1 hr 90 °C incubation (excluding de-paraffination steps)

Elute in ATE Buffer in a final volume of 50 uL

Extraction Methods-SHSU Cadavers

5

• Mock Samples: single buccal swab, pubic hair roots and 3mm x 2mm cuttings

• Automate Express™ PrepFiler™ Express Forensic DNA Extraction Kit

• Digestion

• LySep columns containing PrepFiler Lysis Buffer and 1 M DTT

• Incubation @ 70oC for 40 minutes

• Centrifuge column/tube assembly and transfer lysate

• Purification

• Automate Express with PrepFiler Express cartridges

• Elute in final volume of approximately 40-50 uL

Extraction Methods-Mock Samples

1 2 3 4 5 6 7 8 9 10 11 12Sam

ple Tub

e

Elutio

n Tub

e

0-1

Move to Function Position Numbers

1 2 3 4 5 6 7 8 9 10 11 12Sam

ple Tub

e

Elutio

n Tub

e

0-1

Move to Function Position Numbers

Sample

Tube

Elution

Tube

Magnetic

Particles

Wash

Buffer

x3 Elution

Buffer Isopropanol

Wash

3X

Lysis solution for

BTA protocol

6

• Evaluate DNA quality and quantity with QuantifilerTM Trio DNA

Quantification Kit to make sample processing decisions prior to STR

amplification

• Are quantification results consistent with downstream STR results (expected

recovery and peak heights)?

• Is the Degradation Index (DI) indicative of sample degradation level?

• Does the IPC CT shift, identify the presence of inhibitors that would impact STR

amplification?

• STR Analysis

• GlobalFiler

• Fusion 6C

• Fusion 5C

DNA Quantitative/Qualitative Evaluation and STR Analysis

7

QuantifilerTM Trio DNA Quantification Kit

Maximize accuracy, sensitivity and specificity

Higher Performance with Challenging

Samples

• Improved performance with low-ratio male/female mixture samples

• Short-amplicon and long-amplicon assay targets assess DNA degradation

• IPC optimization to match performance of next generation STR kits

Enhanced Workflow & Data Analysis

• Improved consistency and homogeneity of DNA standard

• Integrated quantification & STR setup tools to support complex casework sample processing

Fast & Accurate Decision Tool

• Shorter time to results (<1 hour)

• Increased assay sensitivity and accuracy

• Additional sample quality info + data review and reaction setup tools = faster, better decisions and results

8

STR Amplification Experimental Design

GlobalFiler Fusion 6 and 5 dye

Thermocycler 9700 9700

Input and cycle 1ng (or less) for 29

cycles

1ng (or less) for 29

cycles

Injection kv and

sec

1.2 kv for 24 sec

(3500xL)

1.2 kv for 24 sec

(3500xL)

ILS/matrix dye LIZ 600/J6 WEN ILS

500/Promega J6


UNT Bone Samples

10

• UNT: extracted and processed 15 bones

• Bone Age Range:

• Contemporary (UNT numbers 1,2,3,8,9,15)

• WWII Era, Historical from the femur of a soldier (UNT10-14)

• Late 19th Century, Historical from Deadwood, SD. From the tooth (UNT4) and

Femur (UNT5-7)

• Trio Results

• No bones exhibited significant inhibition

• DI range from 2 to 51

BONE SAMPLES

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• 3 bones run with all 3 kits

• GlobalFiler

• Fusion 6C with water

• Fusion 5C with water

• All 15 bones run with 2 kits

• GlobalFiler

• Fusion 6C with low TE

• Data Analysis:

• Analyzed with GeneMapper ID-X v1.5

• 175 RFU and 50 RFU for the 3 bone samples

• 50 RFU for all remaining samples

STR Analysis


AT = 175 RFU

3 Bones with GF, F6C and F5C

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Allele Recovery for 3 Contemporary Bones with 3 Kits

0

5

10

15

20

25

30

35

40

UNT2 UNT8 UNT15

Nu

mb

er

of

Allele

d R

eco

vere

d

Alleles Recovered for 20 Core CODIS Loci - 29 cycles, 175 RFU

GF 6C 5C

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Female Bone UNT2: Number of Core Loci alleles recovered

GF: 12

F6C: 10

F5C: 13

vWA under 175 RFU

DI = 22

15

Female Bone UNT8: Number of Core Loci alleles recovered

GF: 14

F6C: 11

F5C: 11

DI = 51

16

• GF 36

• F6C 25

• F5C 17

Male Bone UNT15: Number of Core Loci alleles recovered

GF: 36 F6C: 25 F5C: 17

DI = 10

17

Male bone UNT15 & Male Marker Recovery (175 RFU)

GF Amel, Y indel and DYS391 called

F6C Amel (blue) and DYS391 (purple)

No RM Y’s called

F5C Amel (blue) called

No DYS391 called

x x

x

18

Male bone UNT15 & Male Marker Recovery (50 RFU)

GF Amel, Y indel and DYS391 called

F6C Amel (blue) and DYS391 (purple)

No RM Y’s called

F5C had Amel (blue)

DYS391 at 54RFU

x x


AT = 50 RFU

3 Bones with GF, F6C and F5C

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Allele Recovery for 3 Contemporary Bones with 3 Kits:AT 50RFU

0

5

10

15

20

25

30

35

40

UNT2 UNT8 UNT15

Nu

mb

er

of

Allele

s R

eco

vere

d


GF 6C 5C

DI: 22 51 10

21

UNT 2 at 50 RFU DI = 22

GF: 25 F6C: 18

F5C: 19

Y Axis at 3000 RFU

22

• GlobalFiler - more alleles recovered in the high molecular weight region


Y Axis to 400 RFU

GF: 22

F6C: 16

F5C: 15

23

• GlobalFiler - more alleles recovered in the high molecular weight region


Y Axis to 750 RFU

GF: 38

F6C: 35

F5C: 35


AT = 50 RFU

Core Loci and SE33

(21 Loci total)

15 Bones with GF, F6C (TE)

25

Allele Recovery for 15 bones with GF and F6C with TE

0

5

10

15

20

25

30

35

40

45

UNT1 UNT2 UNT3 UNT4 UNT5 UNT6 UNT7 UNT8 UNT9 UNT10 UNT11 UNT12 UNT13 UNT14 UNT15

Nu

mb

er

of

Allele

s R

eco

vere

d

Alleles Recovered for 21 Loci: GF vs 6C (with TE) - 29 cycles, 50 RFU

GF 6C

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Bones UNT1 (DI = 6) and UNT4 (DI = Undetermined LA)

Y Axis to 255 RFU

UNT1

GF: 19

F6C: 6

UNT4

GF: 20

F6C: 7

370 RFU

27

Bone UNT6—Late 19th Century Femur

Y Axis to 750 RFU

Artifact

DI = 7.6

GF: 31

F6C: 11


AT = 175 RFU and 50 RFU

SHSU: 7 Cadaver Samples with GF, F6C and F5C

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• Cadaver 1

• Male

• 290 days @ 4oC

• Cadaver 2

• Male

• 814 days @ 4oC

• Cadaver 3

• Male

• 113 days @ 4oC

Cadaver information

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Sample

Name

Sample Type/Origin Degradation

Index

IPC

SH1 (Male 1) Heart 451

No significant inhibition observed

SH2 (Male 1) Kidney 219

SH4 (Male 3) Trap Skin 8

SH5 (Male 3) Humerus Marrow 6

SH7 (Male 1) Gluteus Maximus 6

SH8 (Male 2) Psoas Major (long muscle) 24

SH9 (Male 3) Femur Marrow 4

Cadaver samples extracted at Sam Houston State University

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7 Cadaver Samples with 3 kits AT = 175 RFU

0

5

10

15

20

25

30

35

SH1 SH2 SH4 SH5 SH7 SH8 SH9

Nu

mb

er

of

Allele

s R

eco

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d


GF 6C 5C

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7 Cadaver Samples with 3 kits AT = 50 RFU

0

5

10

15

20

25

30

35

SH1 SH2 SH4 SH5 SH7 SH8 SH9

Nu

mb

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of

Allele

s R

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GF 6C 5C

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SH7-Gluteus Maximus DI = 6 Different Axes

Y Axis 4794 RFU GF: 33 Alleles

Y Axis 1865 F6C: 26 Alleles

Y Axis 1377 F5C 21 Alleles

34

SH7-Gluteus Maximus DI = 6 Same Axes

GF: 33 Alleles

F6C: 26Alleles

F5C 21 Alleles

Y Axis 4800 RFU for all kits

35

Y Axis = 2600

SH8-Cadaver long muscle DI = 24

GF 22 Alleles

F6C 17 Alleles

F5C 10 Alleles

36

SH8-Cadaver long muscle DI = 24

Y Axis to 2556 RFUs GF: 22

Y Axis = 605 RFU F6C: 17

Y Axis to 305 RFU F5C: 10


GlobalFiler™, PowerPlex Fusion™ 6C, PowerPlex Fusion 5C

AT = 50 RFU for all 3 kits

Mock Samples—Temperature and Time Studies

38

• Body Fluid Types (All female DNA from same donor)

• Buccal

• Menstrual Blood

• Pulled Pubic Hairs

• Substrates

• Swabs

• Sanitary Napkin

• Cotton material

Body Fluids and Substrates

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• Samples stored outside for 2-12 days in Redland, CA from

13th – 24th April, 2016

• Temperature ranged from 50 to 91 Degrees Fahrenheit

• Humidity up to approximately 77%

• Sample placement

• In plastic bag with wet paper towel outside

• In soil outside

• On table outdoors

• Bathroom window above bathtub

Environmental Conditions

40

• Most samples yielded full profiles with all kits—not degraded.

• Arrows show samples with some level of degradation

Samples run with all 3 kits: GF and Fusion kits (with water)

0

5

10

15

20

25

30

35

40

K2A K2B K5B K7A K7B K10 K12A K14 K16A K16B K17A K17B K18A K18B K20A K20B K21A K21B K22

Nu

mb

er

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Alle

les

Re

cove

red

Allele Count for 20 Core CODIS Loci

GF 6C 5C

41

• Female DNA profile consists of 36 alleles for the

20 CODIS Core Loci comparison: FULL PROFILE WITH GF

Samples K7A (DI = 4) and K7B (DI = 4.7): Weathered Buccal Swabs Outside for 10 days

K7A

GF: 36

F6C:27

F5C: 21

Y Axis to 8185 RFU

Y Axis to 1158 RFU

Y Axis to 876 RFU

42

Sample K7A: Full 36 alleles for GF and 27 for Fusion 6C

CSF 9

TPOX 8, 9

D13 12,14

D7 7,9

D10 13,14

GF Full Profile and PowerPlex Fusion 6C missing 9 Alleles

43

15 Alleles missing from the Fusion 5C sample

Sample K7A: Full 36 alleles for GF and 21 for Fusion 5C

44

Y Axis scaled to each sample separately

Sample K7B

K7B

GF: 36

F6C: 33

F5C: 24

Y Axis to 11,070 RFU

Y Axis to 2834 RFU

Y Axis to 662 RFU

45

All kits = Y Axis set to 10,000 RFU

Sample K7B: GF full profile and much more robust amplification

K7B

GF: 36

F6C: 33

F5C: 24


GlobalFiler™, PowerPlex Fusion™ 6C (Low TE): CODIS Core 20 Loci Allele Recovery

AT = 50 RFU for both kits

Mock Samples--Weathered

47

Samples run with GF and Fusion 6C with Low TE

0

5

10

15

20

25

30

35

40

K3A K3B K5A K6A K6B K8A K8B K9 K11 K12B K15A K15B K19 K23A K23B K24

Nu

mb

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of

Alle

les

Re

cove

red

Allele Count for 20 Core CODIS Loci

GF 6C w/TE

• Most samples yielded full profiles with both kits—not degraded.

• Arrows show samples with some level of degradation

The GOOD

The BAD

and

The

UGLY

?

GONE

48

• Pulled pubic hairs in a plastic bag with wet paper towel outside for 7 days

• Y Axis set to 1200 RFU

K11 (DI = 43.5): The UGLY: Very degraded Sample

GF: 9 alleles

F6C: 4 alleles

49

K11: Did not use Amel or SE33 in CODIS Core Loci count

• Y Axis set to 200 RFU

x

x

SE33 SE33

GF 9 Alleles and PowerPlex Fusion 6C 4 Alleles

50

• Sanitary Napkin with menstrual blood weathered outside for 12 days

• Y Axis set to 125 RFU DI was Undetermined

Sample K23A: ? GONE-Limited intact DNA for amplification

GF: 3 alleles

F6C: No core loci

Amel Only

51

• Dr. Angie Ambers & Dr. Bruce Budowle (UNT)

• Dr. Sheree Hughes-Stamm (SHSU)

• Len Goren (Thermo Fisher Scientific, Global Sales Leader)

• Joshua Abernathy & Jacquelyn Gabriel (Thermo Fisher Scientific/ FAS)

• Kellie Fenesan & Phillip Czar (Thermo Fisher Scientific/ AM)

• Erin McGee, Jarrett Roth & Angela Koenig (Thermo Fisher Scientific/ HPS)

Acknowledgements

52

Joanne B. Sgueglia (Thermo Fisher Scientific, Project Tech Lead)

Questions:

[email protected]

Thank You


For Forensic & Paternity Use only. Not for use in diagnostic procedures. ©2016 Thermo Fisher Scientific Inc. All Rights Reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Fusion is a trademark of Promega. QIAamp is a trademark of Qiagen.

54

• Some GF comparisons were run with TE for amp set up of Fusion 6C

• Comparison of same samples run with water and TE for Fusion 6C

• Total of 31 samples run at 175 RFU and 50 RFU

• UNT 2, 8, 15

• SH1- SH9

• K2A, K2B, K5B, K7A, K7B, K10, K12A, K14, K16A, K16B, K17A, K17B, K18A,

K18B, K20A, K20B, K21A, K21B, K22

• Conclusions

• Bone samples slightly better with water

• Cadaver samples significantly better with TE

• Mock samples significantly better with TE

Fusion 6C and comparison of water vs. TE for Amp Set Up

55

Sample Quant (SA) <1ng DI

Others > 1ng In range of 2-10 DI > 10 DI

K4 0.000493

Und LA

K6A 0.011549

1.558924

K6B 0.017837

1.525564

K8A 0.29501

6.820662

K8B 0.361047

3.61877

K10 0.686702

1.970416

K11 0.009714

43.53462

K15A 0.209954

0.964699

K15B 0.019852

3.599267

K17A 0.733631

2.893576

K20A 0.6447

2.62813

K20B 0.770973

2.686473

K23A undetermined und

56

Samples with > 1ng and DI > 2

Sample DI In range of 2-10 DI

K7A

3.937109

K7B

4.658913

K16A

2.05177

K17B

3.094319

K21B

2.085674

2016 degradation study - thermo fisher scientific - …...3 •unt: modified organic method •24...

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