2016 degradation study - thermo fisher scientific - …...3 •unt: modified organic method •24...
TRANSCRIPT
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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
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UNT Bone Samples
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• 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
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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
14
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
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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
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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
Alleles Recovered for 20 Core CODIS Loci - 29 cycles, 50 RFU
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
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• GlobalFiler - more alleles recovered in the high molecular weight region
UNT 8 at 50 RFU DI = 51
Y Axis to 400 RFU
GF: 22
F6C: 16
F5C: 15
23
• GlobalFiler - more alleles recovered in the high molecular weight region
UNT 15 at 50 RFU DI = 10
Y Axis to 750 RFU
GF: 38
F6C: 35
F5C: 35
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AT = 50 RFU
Core Loci and SE33
(21 Loci total)
15 Bones with GF, F6C (TE)
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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
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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
vere
d
Alleles Recovered for 20 Core CODIS Loci - 29 cycles, 175 RFU
GF 6C 5C
32
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
er
of
Allele
s R
eco
vere
d
Alleles Recovered for 20 Core CODIS Loci - 29 cycles, 50 RFU
GF 6C 5C
33
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
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SH7-Gluteus Maximus DI = 6 Same Axes
GF: 33 Alleles
F6C: 26Alleles
F5C 21 Alleles
Y Axis 4800 RFU for all kits
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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
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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
of
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
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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
er
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:
Thank You
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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