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Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
Maximizing Allele Detection by Selecting
Optimal Analytical Thresholds
Christine A. Rakay
Joli Bregu
Cheng-Tsung Hu
Catherine M. Grgicak
American Academy of
Forensic Sciences February
2013
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
Steps During DNA Interpretation
Comparison to
Known(s)
Validation
studies &
Literature
Application of
Thresholds
Effect of AT’s on Data Analysis
2 males, 1:19 at 2ng, 1ul 3130 prep volume and 5s injection
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
Summary of Methods Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
ISHI Mixture Interpretation Workshop, 2012. http://www.cstl.nist.gov/strbase/mixture.htm
89% of respondents use an AT between
50 and 150 RFU
Summary of Methods
Method 1.
◦ Kaiser (IUPAC 1976)
Winefordner 1983 and Krane 2007
Method 2.
◦ Currie (IUPAC 1995)
Winefordner 1983
Method 3.
◦ Example in SWGDAM Guidelines
Method 4.
◦ Largest observed noise peak
Method 5.
◦ Miller & Miller. Statistics for Analytical Chemistry (Ellis Horwood & Prentice Hall)
IUPAC 1997 ElectroAnalytical Committee
Method 6.
◦ 1997 IUPAC ElectroAnalytical Committee Recommendations
Use d
ata
fro
m n
egatives (
i.e.
sam
ple
s w
ith n
o D
NA
)
Use d
ata
fro
m
DN
A d
ilution
series
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
J. Bregu et al. Analytical Thresholds and Sensitivity: Establishing RFU Thresholds for Forensic DNA Analysis. JFS (2013) 1
pg 120-129.
Method 1 to 4 - Negatives Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
-Negative sample run with an internal size standard (not shown) using manufacturer’s
recommended protocol
Negative = extraction or amplification negative
15
0
15
0
15
0
15
0
Green and Blue channels seem ‘quieter’ than yellow and red
Baseline is never below 0 RFU Processed data!
Method 5 to 6 – Positives (Standard Curves) Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
Regression of positive samples (i.e. single source samples)
Amplified 0.0625-4ng dilution series, injected 5s using manufacturer’s recommended protocol
Plot of Input DNA (ng) versus average peak height (per color) – with error bars
◦ If a peak was homozygous, the RFU was divided by 2
• The points at 2 and 4 ng fall off
the line (PCR efficiency approaching a plateau)
• The error bars become larger with increased DNA input
• A weighted linear regression is within the linear range (i.e. 0.0625 – 1 ng) was used.
Summary of Results
Method Origin
Analytical Threshold for green
5s injection example
1 Negatives 7
2 Negatives 4
3 Negatives 18
4 Negatives 6
5 DNA Series 31
6 DNA Series 39
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
False non-labeling of alleles (Drop-out) Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
Single source 0.125ng, 1ul 3130 prep volume
0
200
40
20
80
Drop-out with Respect to ATs - <0.5 ng DNA Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
0
0.2
0.4
0.6
0.8
1
0 50 100 150 200
Fre
qu
en
cy o
f Dro
p-o
ut
Analytical Threhold (RFU)
■ locus DO
■ allelic DO
■ sum (# loci exhibiting
DO)
-As AT increases, locus DO increases, while allele DO stabilizes after 50 RFU then starts to decrease after AT of ~150 RFU. -Although a higher AT (i.e. >150 RFU) begins to decrease the number of loci where allele DO occurs (less stochastic variation),
-Locus DO increases, resulting in an overall increase in DO with AT for Low-template samples
hetlocitotal
alleleDOhetlocilocusfreqDO
#
)2(#)(
hetlocitotal
alleleDOhetlociallelefreqDO
#
)1(#)(
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
-AT’s have a large effect on the ability to detect/label alleles.
-Red = high level of allele drop-out, blue=low levels of allele drop-out. - To take a ‘conservative’ approach and utilize high AT values leads to a substantial level of Type II errors for low-level samples (i.e. <1000RFU).
Balancing Type I and Type II Errors – < 0.5ng
Impact of ATs on STs Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
Analytical
Threshold
(RFU)
Description of
Calculation
Frequency of
Allele Drop-
out
Peak Height of Largest
Surviving Allele at a locus
exhibiting allele drop-out
(RFU)
Frequency of
Locus Drop-
out
Negatives Kaiser’s 0.025 126 0.000
Negatives Kaiser’s 0.041 126 0.002
Negatives Max observed
noise peak 0.168 170 0.028
Positives IUPAC 0.230 240 0.074
50 N/A 0.246 229 0.074
150 N/A 0.246 349 0.593
200 N/A 0.184 548 0.816
N/A = not applicable
500 – 63 pg
Baselines Positives ≠ Baselines Negatives
30
0
30
0
30
0
High
input of
DNA
Neg amp
control
Low
input of
DNA
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
More on Baselines and Noise Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
• This is not instrument baseline/noise
• Single source DNA data amplified from 0.0625 – 2 ng
• Differentiated ‘noise’ from artifact
• -A, pull-up, stutter (+ or -), spikes, dye artifacts
• Plotted RFU of the known/expected peak versus the highest ‘noise’ peak
• High noise with >0.5 ng of DNA, higher AT needed for higher-template samples
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.0 0.1 0.2 0.3 0.4
Pro
po
rtio
n o
f m
ino
r al
lele
lab
led
Proportion of Loci with Noise >AT
Mixture 1:9
> 0.5 ng
<0.5 ng
ATM2
AT= 50
Injection Times Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
0
20
40
60
80
100
120
<500 500-1000 1000-1500 1500-2000 2000-2500
Nu
mb
er o
f p
eaks
Avg allele height (RFU)
Amplification positive sample (2 sec)
blue
green
yellow
red
020406080
100120140160180200
Nu
mb
er
of
pe
aks
Avg allele height (RFU)
Amplification positive samples (5 sec)
blue
green
yellow
red
020406080
100120140160180200
<5
00
50
0-1
00
0
10
00
-15
00
15
00
-20
00
20
00
-25
00
25
00
-30
00
30
00
-35
00
35
00
-40
00
40
00
-45
00
45
00
-50
00
50
00
-55
00
55
00
-60
00
60
00
-65
00
65
00
-70
00
70
00
-75
00
Nu
mb
er o
f p
eaks
Avg allele height (RFU)
Amplification positive samples (10 sec)
blue
green
yellow
red
injection times
# of noise peaks
height noise peaks
AT
High-template Low-template
injection times
# of noise peaks
height noise peaks
AT
Conclusions
• Baseline does not remain constant between negatives and samples
with a significant amount of DNA
• There may be amplification ‘noise’ that cannot be characterized as
known artifact (i.e. bleed-through, spike, stutter, etc)
• Optimal ATs will be dependent on the DNA amplification mass
• Optimal AT for DNA samples amplified with < 0.5 ng was 10- 20
RFU.
• An AT of 50 resulted in ~ 20% Type II error rate. An AT of
150 resulted in ~80% error rate.
• To minimize error for DNA samples amplified with > 0.5 ng the AT
needs to be increased by a factor of 2.5 - 5 (i.e. 50 RFU)
• Thresholds designed for/by samples containing optimal masses are not
optimal for low-template DNA interpretation
• Samples amplified with sub-optimal masses require special
interpretation schemes/methods
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118
Acknowledgements
Thanks to the following Boston University BMFS students,
◦ Christine A. Rakay
◦ Joli Bregu
◦ Kevin Hu
◦ Thank-you
◦ Robin Cotton, Charlotte Word, Michael Coble, John Butler, Desmond Lun
◦ Supported by
◦ NIJ2008-DN-BX-K158 training grant awarded by the National Institute of Justice, Office
of Justice Programs, U.S. Department of Justice. The opinions, findings and
conclusions or recommendations expressed in this presentation are those of the
authors and do not necessarily reflect those of the Department of Justice
◦ Boston University, Biomedical Forensic Sciences Program
Boston University School of Medicine
Program in Biomedical Forensic Sciences
72 E. Concord Street, Boston, MA 02118