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TRANSCRIPT
In-field Collection of DNA from Decomposing
Remains for More Direct Analysis: Experiences with Investigator Quality Sensors
Amy S. Holmes, PhD*; Ryan Gutierrez, BS; Madeline Roman, BS;
David Gangitano, PhD; Sheree Hughes-Stamm, PhD
Department of Forensic Science
Sam Houston State University
Huntsville, TX USA
7th QIAGEN Investigator Forum, May 2018;
San Antonio, TX USA
This project was partially funded by a Graduate Research Fellowship Award #2015-R2-CX-0029 (National
Institute of Justice, Office of Justice Programs, U.S. Department of Justice).
The opinion, findings, conclusions, or recommendations expressed in this presentation are those of the authors
and do not necessarily reflect those of the National Institute of Justice.
ROLE OF FUNDING
Please do NOT take any photographs of slides
depicting human remains
Disaster Victim Identification (DVI)Mass fatalities
Remote locations
High temperatures & humidity
Limited/lack of facilities
Loss of electricity
Inability to perform DNA analysis for weeks/month
Skeletal samples
GoalsFast, simple DNA collection (in field)
First 2 weeks (or prior to skeletonization)
RT storage of samples
Faster sample processing for STR typing
2004 SE Asian Tsunami >220,000 deceased
Days of Decomposition
Day 1 Day 7 Day 14
April – Average Temp 15 – 25ºC
Day 0
Day 4
Day 6
Day 10
Day 14
QS1 QS2Investigator 24plex QS – 100% SuccessInvestigator 24plex QS – 100% Success
Sampling Approach - Swabs
Skin
Muscle
Processed immediately or stored for 1 month
Traditional
Direct
Investigator® STR GO! Lysis Buffer
QIAamp® DNA
Investigator kit
Investigator
Quantiplex® Pro
Whatman® EasiCollect™
Foam
NylonCotton
0
20
40
60
80
100
Skin Muscle Skin Muscle Skin Muscle
Cotton Foam NylonA
llele
s R
epo
rted
(%
)Fresh Tissue
STR Results - Swabs
Direct PCR – GO!Traditional Analysis – QS
0
20
40
60
80
100
Skin Muscle Skin Muscle Skin Muscle
Cotton Foam Nylon
Alle
les
Rep
ort
ed (
%)
0
20
40
60
80
100
Skin Muscle Skin Muscle Skin Muscle
Cotton Foam NylonA
llele
s R
epo
rted
(%
)
Fresh Tissue Decomposed Tissue
• Full profiles with fresh tissues (skin & muscle)
• Cotton swabs & the foam heads from Whatman® EasiCollectTM Devices seemed to generate the most
consistent results regardless of the tissue type sampled
• Direct PCR of swabs less successful from decomposed remains; Nylon – PCR inhibition, dilution required
0
20
40
60
80
100
Skin Muscle Skin Muscle Skin Muscle
Cotton Foam Nylon
Alle
les
Rep
ort
ed (
%)
Fresh Tissue Decomposed Tissue
QS
Inhibition – Nylon Swabs
GO!
Small Amplicon 0.0487
Large Amplicon 0.0000
Degradation I n/a
IPC -0.31
Allele % 100
Small Amplicon 0.0084
Large Amplicon 0.0000
Degradation I n/a
IPC -13.87
Allele % 0
• Other direct STR kits?
• No DNA, PCR failure, and/or complete inhibition?
Sample NameSample Name SQOSQO SOSSOS SQSQ SSPKSSPK MIXMIX OMROMR CGQCGQ
GeneMapper® ID-X 1.4
Project: Amy PhD Samples pt 1-dilutions 112216
Wed Mar 07,2018 10:39AM, CST Printed by: gmidx Page 1 of 1
D1T0 DPD1T0 DP
… TH01 D3S1358 vWA D21S11
Y1585
9.31621
161587
32.21740
X1477
71788
153664
142212
292150
0
1900
60 120 180 240 300 360 420 480
D1T0 DPD1T0 DP
TPOX DYS391 D1S1656 D12S391 SE33
111293
161746
24791
20.21572
82189
111611
152027
21910
191427
0
1900
60 120 180 240 300 360 420 480
D1T0 DPD1T0 DP
D10S1248 D22S1045 D19S433 D8S1179 D2S1338
151524
151035
20851
142099
163268
121097
132913
17914
0
1900
60 120 180 240 300 360 420 480
D1T0 DPD1T0 DP
D2S441 D18S51 FGA
161277
201581
101201
141710
141124
191209
0
1900
60 120 180 240 300 360 420 480
D1T0 DPD1T0 DP
… D16S539 CSF1PO D13S317 D5S818 D7S820 …
111715
121209
Q1407
101850
111176
91678
141336
122457
81436
121282
S2904
0
1900
60 120 180 240 300 360 420 480
Dilution
98% Alleles
93% Alleles
29% Alleles
Co
tto
nN
ylo
nF
oam
DECOMPOSED
QS
GO!
QS
GO!
QS
GO!
100% Alleles
93% Alleles
81% Alleles
QS1 QS2
Preservation – 1 month
• No significant difference between skin & muscle tissue
• Significantly fewer alleles were recovered from samples collected & stored on Nylon
4N6FLOQSwabs for 1 month
0
20
40
60
80
100
Cotton Foam Nylon Cotton Foam Nylon
Skin Muscle
Alle
les R
eport
ed (
%)
Decomposed Tissues
Time 0 1 Month
Skin Muscle
Quantifiler Trio indicates low to moderate degradation, no inhibition
Quantiplex Pro indicates high degradation and no inhibition
ST: 0.18LT: 0.054DI: 3.26
IPC: -0.5798% Alleles
ST: 0.24LT: 0.011DI: 21.6
IPC: -0.3498% Alleles
Quantifiler Trio &
GlobalFiler
QuantiplexPro &
24plex QS
Confirmed by QS
Examples of Reworked Samples
100% Alleles
24% Alleles
Other direct PCR kits - Extreme degradation, inhibition?
Examples of Reworked Samples
Dilution
GO!
Partial Profile
100% Alleles
69% Alleles
Dilution
Examples of Reworked Samples
QS imbalance
GO!
Partial Profile
Sampling Approach - Biopsy
Modified TENT Buffer10mM Tris, 10mM EDTA, 1M NaCl, 2% Tween 20
(DNA leaches into solution)
Stored for up to 1, 3, & 6 months
PDQeX (ZyGEM) QIAquick 1:10 Dilution (no purification)
Quantifiler Trio
24plex QS
24plex GO!
2μL
<20 min <30 min
Quantification Results
0
1
2
3
4
5
6
0
40
80
120
160
200
Fresh Early Decomposition AdvancedDecomposition
Deg
rad
atio
n In
dex
DN
A C
on
cen
trat
ion
(n
g/µ
L)
Small Amplicon Quantification Degradation Index
Days 0-4 Days 6-8 Days 10-14
0
10
20
30
40
50
60
70
80
90
100
PDQex Tissue PDQex TENT Qiaquick Tent1:10 QS Tent 1:10 Go
Avera
ge N
o. A
llele
s (
%)
Fresh Early Advanced
Days 0-4 Days 6-8 Days 10-14
STR Results
QIAquick
TENT
TENT 1:10
QS
PDQeX
TENT
PDQeX
Tissue
FRESH TISSUES – 6 month storage
TENT 1:10
GO!
100% Alleles
100% Alleles
100% Alleles
100% Alleles
100% Alleles
Early Decomposition (days 6 – 8)
Before/onset of Bloat
Differences in Decomposition
QIAquick
TENT
TENT 1:10
QS
PDQeX
TENT
PDQeX
Tissue
TENT 1:10
GO!
98% Alleles
100% Alleles
89% Alleles
84% Alleles
100% Alleles
DECOMPOSED – Before/Onset of Bloat
QS1 QS2
Advanced Decomposition (days 10 - 14)
Difference in Decomposition
QIAquick
TENT
TENT 1:10
QS
PDQeX
TENT
PDQeX
Tissue
TENT 1:10
GO!
98% Alleles
99% Alleles
80% Alleles
82% Alleles
100% Alleles
ADVANCED DECOMPOSITION
87% Alleles
20% Alleles
QIAquick
TENT
TENT 1:10
QS
PDQeX
TENT
PDQeX
Tissue
TENT 1:10
GO!
60% Alleles
42% Alleles
22% Alleles
ADVANCED DECOMPOSITION
Low/No DNA present
GO!
No/little benefit in re-running sample in attempt to improve profile
Re-extract?
Small Amplicon 0.0001
Large Amplicon 0.0000
Degradation N/A
IPC 0.049
QS
Highly Degraded
GO!
Small Amplicon 8.2841
Large Amplicon 0.0066
Degradation I 1255.17
IPC 0.12
Allele % 75.56
Highly degraded, No inhibition detected
Re-run based on LA or another approach?
84% Alleles
Sample NameSample Name SQOSQO SOSSOS SQSQ SSPKSSPK MIXMIX OMROMR CGQCGQ
GeneMapper® ID-X 1.4
Project: AS New Swabs Study Reamp 2 022618 RMG
Wed Mar 07,2018 10:54AM, CST Printed by: gmidx Page 1 of 1
E0F4M qsE0F4M qs
… TH01 D3S1358 vWA D21S11
Y 9.3 16 32.2
X OL 8 17 14 30
0
16000
60 120 180 240 300 360 420 480
E0F4M qsE0F4M qs
TPOX DYS391 D1S1656 D12S391 SE33
OL
12
OL OL 15.3 17
OL 8 OL 10 12 15 15 20 16.2
0
17000
60 120 180 240 300 360 420 480
E0F4M qsE0F4M qs
D10S1248 D22S1045 D19S433 D8S1179 D2S1338
15 15 13 20
OL 14 17 11 16 14 12 18
0
14000
60 120 180 240 300 360 420 480
E0F4M qsE0F4M qs
D2S441 D18S51 FGA
OLSpike20745
OL OL 24
11 14 OL 15 18 22
0
13000
60 120 180 240 300 360 420 480
E0F4M qsE0F4M qs
… D16S539 CSF1PO D13S317 D5S818 D7S820 …
OL
S 12
Q OL 11 11 14 8 11 12 9 S
0
15000
60 120 180 240 300 360 420 480
Large Target
Sample NameSample Name SQOSQO SOSSOS SQSQ SSPKSSPK MIXMIX OMROMR CGQCGQ
GeneMapper® ID-X 1.4
Project: AS New Average Amp 022818 RMG
Wed Mar 07,2018 01:34PM, CST Printed by: gmidx Page 1 of 1
E0F4M QSE0F4M QS
… TH01 D3S1358 vWA D21S11
Y7897
16154
X4572
9.313807
174554
14304
0
9000
60 120 180 240 300 360 420 480
E0F4M QSE0F4M QS
TPOX DYS391 D1S1656 D12S391 SE33
1216805
15815
814763
103702
12799
15570
20486
17206
0
9000
60 120 180 240 300 360 420 480
E0F4M QSE0F4M QS
D10S1248 D22S1045 D19S433 D8S1179 D2S1338
178216
15936
1513270
11733
161145
14345
0
9000
60 120 180 240 300 360 420 480
E0F4M QSE0F4M QS
D2S441 D18S51 FGA
143256
182080
117158
151859
24122
0
9000
60 120 180 240 300 360 420 480
E0F4M QSE0F4M QS
D16S539 CSF1PO D13S317 D5S818 D7S820 ……
141354
11191
127628
111446
8160
12180
S1714
Q979
0
9000
60 120 180 240 300 360 420 480
Average of
Small & Large
Targets
• All methods preserved DNA well for relatively fresh tissues (up to 4 days in this
study)
• Most promising methods for collecting & storing DNA at RT from decomposed
remains:
• Cotton or foam swabs via incision in underlying muscle tissue
• Tissue biopsy in TENT buffer rapidly purified with PDQeX 2400 System
• Direct PCR approaches show some potential but require optimization
• Quality indicators in the Quantiplex Pro kit can increase first-pass success rates,
and Quality Sensors in the Investigator 24plex STR kits can be used to confirm
best route for sample rework (or not)
Conclusions
Acknowledgements
• Department of Forensic Science, Sam Houston State University
• PhD Advisor: Dr. Sheree Hughes-Stamm
• Graduate Students: Carrie Mayes, Kyleen Elwick, Michelle Harrel, Esiri “Ace” Tasker, Ryan Gutierrez, Maddie Roman
Acknowledgements
• QIAGEN® for support. Special thanks to Keith Elliot, Dr. Meredith Turnbough, Bryan Davis, Angela Cacioppo, Inga Gerard
• NIJ GRF Funding #NIJ 2015-R2-CX-0029
• Applied Anatomical Research Center (AARC)
• Donors and families of donors
Amy Holmes, PhD
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