evaluating stochastic thresholds for six amplification...
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© 2011 The Bode Technology Group, Inc.
Evaluating Stochastic Thresholds
for Six Amplification Kits
Presented by: Stephanie Sivak
© 2011 The Bode Technology Group, Inc.
Outline
• Study performed by Bode in conjunction
with VCU
• Purpose
• Stochastic drop out
• Study design
• Results
© 2011 The Bode Technology Group, Inc.
Stochastic drop-out
• Defined in this study:
• An event where a pair of alleles exhibit one allele above an established analytical threshold and one allele below – Sister allele could also
drop out completely
75RFU
© 2011 The Bode Technology Group, Inc.
Stochastic drop-out
• May be affected by a number of
contributing factors in the laboratory
• Amplification conditions: template input,
reaction volume and cycle number
• Sequencing conditions: injection time and
voltage
© 2011 The Bode Technology Group, Inc.
Purpose
• Problem: Analyst variability with mixture
interpretation
• NIST Mix 05 Study
– Confirmed the issue
© 2011 The Bode Technology Group, Inc.
Purpose
• Baby Steps: Stochastic threshold
evaluation
• Original validation studies- homozygote
threshold
• Increased sensitivity
– Type of samples testing
– Optimization techniques
© 2011 The Bode Technology Group, Inc.
Purpose
• 2009 FBI/QAS requirement to set stochastic threshold – “The laboratory establishes a stochastic threshold based on
empirical data derived within the laboratory and specific to the quantitation and amplification systems (e.g., kits) and the detection instrumentation used.”
• 2010 SWGDAM requirement to set stochastic threshold – “Internal validation studies conducted after the date of this
revision shall include as applicable: database-type samples, reproducibility and precision, sensitivity and stochastic studies, and contamination assessment. Internal validation studies shall be documented and summarized. The technical leader shall approve the internal validation studies.”
© 2011 The Bode Technology Group, Inc.
Experimental Design
• Evaluate single source and mixture samples
– First 4 amplification kits: P+, CO, ID, PP16
• Use of single source
– Amp at different low level concentrations
– Determine a peak detection threshold
– Evaluate analysis threshold
– Suggestions for stochastic thresholds
• Does the introduction of more than one person’s DNA affect stochastic threshold
© 2011 The Bode Technology Group, Inc.
Experimental design
• Evaluate mixture profiles at levels where
interpretation is difficult due to the
possibility of drop out
• Based on this data make
recommendations for a stochastic
threshold that would aid in interpretation
© 2011 The Bode Technology Group, Inc.
Experimental Design
• Evaluated 75 potential donors
– Consent
– Level of heterozygosity
– Rarity of alleles in profile
– Gender
• Initial studies based on individual displaying the most heterozygosity
• Chose 13 donors to create 9 two-person mixture samples
© 2011 The Bode Technology Group, Inc.
Experimental Design Chosen donor alleles:
© 2011 The Bode Technology Group, Inc.
Experiment Design
• Peak Detection
– Level of DNA needed to generate detectable results from low template samples
– Targeted: 50pg, 100pg, 150pg, 200pg
– Amplified with 4 amplification kits • Profiler Plus
• COfiler
• Identifiler
• PowerPlex 16
© 2011 The Bode Technology Group, Inc.
• Samples were extracted on an EZ1 and
quantified with Quantifiler
• All samples were amplified in 25ul reaction
volumes
• P+,CO, ID = 28 cycles
• PP16= 30 cycles
• All samples were injected at (3kV 10s)
Methods
© 2011 The Bode Technology Group, Inc.
• Analyze data at the baseline- 1 RFU
• Any peaks attributed to real data deleted
• Calculate average peak height for
remaining data points (noise)
• x 3 standard deviations
Method-Peak Detection
© 2011 The Bode Technology Group, Inc.
Results-Peak Detection
Amplification Kit Peak Detection
Profiler Plus ID 20 RFU
COfiler 20 RFU
Identifiler 25 RFU
PowerPlex 16 30 RFU
• Minimum value we can start to determine real
data from background noise
• Minimum value to look for drop out peaks
© 2011 The Bode Technology Group, Inc.
Stochastic Threshold
• Evaluation proposed in different ways
– Logistic regression
– Above highest peak in the most severe
imbalance
– Plotting PHR v Avg RFU
© 2011 The Bode Technology Group, Inc.
Sources
• Gill, Peter et al. FSI: Genetics 3 (2009) 104-111
• Puch-Solis R. et al. FSI: Genetics Supplement Series 2 (2009) 460-461
• Bright Jo-Anne et al. FSI: Genetics 4 (2010) 111-114
• Tvedebrink Torben et al. FSI: Genetics 3 (2009) 222-226
• Balding, David J and Buckleton, John. FSI: Genetics 4 (2009) 1-10
• Budowle B. et al. J Forensic Sci 54(4)(2009) 810–821
• NIST presentations
• User Forums
© 2011 The Bode Technology Group, Inc.
PHR v Avg RFU
NIST presentation- how to determine the stochastic threshold
• Examine intensity and peak height ratios of samples at different low level concentrations
• Observe variation in peak height ratio and peak intensity
• The stochastic threshold is the point at which variation begins a rapid increase
© 2011 The Bode Technology Group, Inc.
Results- Peak Height Ratio
vs RFU
• At which RFU value does the majority of the peak height ratios fall below our expected balance – P+,CO, ID= 60%
– PP16= 50%
– Using an analytical threshold of 75 RFU
• Plotted PHR v Avg RFU for 50, 100, 150, 200pg
• Zoom in on PHR below expected balance % – Heights could be considered unreliable under this
value and drop out may be possible
© 2011 The Bode Technology Group, Inc.
Comparison of Signal Strength with Peak Height Ratio for Profiler
Plus 25ul Reactions at 28 cycles using 50, 100, 150 and 200pg of
Template DNA
0
20
40
60
80
100
120
0 50 100 150 200 250 300 350
Average RFU per Allele
Peak H
eig
ht
Rati
o (
sh
ort
er
peak/t
all
er
peak)
© 2011 The Bode Technology Group, Inc.
Comparison of Signal Strength with Peak Height Ratio for COfiler
25ul Reactions at 28 cycles using 50, 100, 150 and 200pg of Template
0
20
40
60
80
100
120
0 100 200 300 400 500
Average RFU per Allele
Peak H
eig
ht
Rati
o (
Sh
ort
er
peak/t
all
er
peak)
© 2011 The Bode Technology Group, Inc.
Comparison of Signal Strength with Peak Height Ratio for Identifiler
25ul Reactions at 28 cycles using 50, 100, 150 and 200pg of Template
DNA
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
0 50 100 150 200 250 300 350 400
Average RFU per Allele
Peak H
eig
ht
Rati
o (
sh
ort
er
peak/t
all
er
peak)
© 2011 The Bode Technology Group, Inc.
Comparison of Signal Strength with Peak height Ratio for
PowerPlex 16 25ul Reactions at 30 cycles using 50, 100, 150 and
200pg of Template DNA
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
0 100 200 300 400 500 600 700 800 900
Average RFU per Allele
Peak H
eig
ht
Rati
o (
sh
ort
er
peak/t
all
er
peak)
© 2011 The Bode Technology Group, Inc.
• Based on one sample
– Triplicate amplifications at 4 concentrations
– May be able to lower for ID
– Suggest increasing above 200 rfu for PP16
• Determine targeted amounts for mixture study
• What influences stochastic thresholds?
– DNA quantity?
– Multiple contributors?
Summary
© 2011 The Bode Technology Group, Inc.
Mixture Study • Target amount of minor contributor for
mixture study
– PP16
• 100pg
• 75pg 50:50
– ID, P+, CO
• 150pg
• 100pg 50:50
© 2011 The Bode Technology Group, Inc.
Experiment Design
• Two-person mixtures
– Paired 7 females with 6 males to make 9 two-person mixtures
© 2011 The Bode Technology Group, Inc.
Experiment Design
• Different ratios F:M
• 8 ratio
comparison/pair
• 9 pairs
• 3 amplifications/ea
• 4 amplification kits
• 864 total profiles
Donor Ratio Concentration (pg)
Female 95% 2850
Male 5% 150
Female 90% 1350
Male 10% 150
Female 80% 600
Male 20% 150
Female 50% 100
Male 50% 100
Female 50% 150
Male 50% 150
Female 20% 150
Male 80% 600
Female 10% 150
Male 90% 1350
Female 5% 150
Male 95% 2850
© 2011 The Bode Technology Group, Inc.
Experiment Design
• Each profile evaluated to determine threshold above highest peak in most severe imbalance allele pair – 1 allele ≥150RFU and the other allele is between 20-
74RFU
– Determine if we dropped our analytical threshold could we drop our stochastic
• Plotted PHR of minor contributor and major contributor vs RFU
© 2011 The Bode Technology Group, Inc.
Profiler Plus Results Profiler Plus™
Sample Name Locus Peak Height 1 Peak Height 2
F1_50_M1_50_100pg FGA 38 153
F1_50_M1_50_100pg D18S51 48 165
F6_50_M1_50_150pg D18S51 51 155
F3_50_M3_50_150pg vWA 53 203
F5_50_M5_50_150pg D7S820 55 172
F3_80_M3_20 D21S11 55 210
F2_50_M2_50_100pg D18S51 55 240
F6_50_M1_50_150pg D21S11 58 173
F1_50_M1_50_150pg vWA 59 209
F1_50_M2_50_150pg FGA 61 166
F7_50_M6_50_150pg D5S818 65 152
F1_10_M1_90 D18S51 68 154
F1_5_M1_95 D18S51 69 211
© 2011 The Bode Technology Group, Inc.
COfiler Results
COfiler™
Sample Name Locus Peak Height 1 Peak Height 2
F2_50_M3_50_150pg D16S539 24 176
F2_90_M2_10 TPOX 48 185
F2_50_M2_50_100pg D16S539 49 156
F2_95_M2_5 TPOX 50 155
F1_20_M1_80 D3S1358 51 167
F2_50_M2_50_150pg D16S539 55 158
F1_50_M2_50_150pg D16S539 62 240
F7_50_M6_50_100pg D16S539 71 217
F3_20_M3_80_CO D16S539 74 157
© 2011 The Bode Technology Group, Inc.
Identifiler Results Identifiler™
Sample Name Locus Peak Height 1 Peak Height 2
F7_50_M6_50_150pg D16S539 43 164
F1_50_M2_50_150pg D21S11 43 172
F3_50_M3_50_150pg CSF1PO 44 180
F4_20_M4_80 TPOX 49 179
F1_20_M2_80 D16S539 50 173
F5_50_M5_50 D16S539 58 176
F2_50_M3_50 D8S1179 59 200
F4_80_M4_20 D19S433 60 166
F5_50_M5_50_150pg D16S539 62 172
F4_50_M4_50_100pg TPOX 66 172
F5_50_M5_50_150pg CSF1PO 67 156
F2_80_M2_20 FGA 68 153
F3_50_M3_50_150pg CSF1PO 70 222
F1_10_M2_90 D16S539 70 234
© 2011 The Bode Technology Group, Inc.
PowerPlex® 16
Sample Name Locus Peak Height 1 Peak Height 2
F2_10_M2_90 D18S51 35 222
F2_50_M2_50_75pg D16S539 40 236
F2_50_M2_50_100pg D16S539 42 209
F1_50_M2_50_100pg D5S818 43 184
F1_50_M1_50_75pg D7S820 43 161
F2_80_M2_20 Penta E 44 247
F1_50_M2_50_100pg D16S539 44 251
F1_80_M1_20 Penta D 45 178
F2_50_M2_50_75pg D16S539 46 195
F2_50_M2_50_75pg D18S51 50 184
F2_95_M2_5 TPOX 51 212
F1_50_M1_50_75pg D18S51 59 154
F3_5_M3_95 Penta E 60 210
F6_50_M1_50_75pg Penta E 60 156
F1_50_M2_50_75pg D16S539 60 168
F2_50_M2_50_100pg Penta E 65 155
F1_50_M2_50_75pg Penta E 65 266
F3_20_M3_80 D21S11 66 179
F1_20_M2_80 FGA 66 167
F6_50_M1_50_75pg D18S51 72 193
F2_20_M2_80 Penta E 72 179
F2_5_M2_95 Penta E 74 184
© 2011 The Bode Technology Group, Inc.
Summary • ABI kits appeared consistent between each other but the
stochastic variation depending on the analytical
threshold – 160RFU - 240RFU
• Promega PP16 kit produced the most stochastic
occurrences but the stochastic variation was more
consistent and not as dependent on the analytical
threshold – 250RFU - 260RFU
• Did not want to implement so many different thresholds
or rely on outlier data – Cannot account for every genetic anomaly
© 2011 The Bode Technology Group, Inc.
Results Heterozygous Balance for Identifiler - 28 cycles, 25 ul reactions
Run on ABI 3100 Sequencer
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
0 100 200 300 400 500 600 700
RFU per Allele
He
tero
zy
go
us
Ba
lan
ce
(%
)
© 2011 The Bode Technology Group, Inc.
Summary • Began implementing use of stochastic
thresholds in mixture interpretation in 2009.
• If a locus exhibits alleles below the stochastic threshold then drop out may be possible
– Increase in inconclusive results
– Analysts consistency greatly improved
© 2011 The Bode Technology Group, Inc.
Recommendations
• Single source profile with high level of
heterozygosity
• Get data points from ~3-5 individuals
• Plot PHR vs Avg RFU
Evaluation of Identifiler Plus
and PowerPlex 16 HS
• Evaluated new Identifiler Plus and
PowerPlex 16 HS amp kits
– Prior to manufacture, companies stated these
kits would provide greater sensitivity and
improve mixture interpretation
• Better balance = lower analytical threshold
© 2011 The Bode Technology Group, Inc.
© 2011 The Bode Technology Group, Inc.
Experimental Design
• Evaluate single source samples only
– ID+ and PP16 HS
• Amp at different low level concentrations
• Plot PHR vs Avg RFU
– Evaluate analysis thresholds
– Suggest stochastic thresholds
© 2011 The Bode Technology Group, Inc.
• Samples previously extracted on EZ1s
and quantified with Quantifiler
• All samples were amplified in 25ul reaction
volumes
• ID += 28 cycles
• PP16 HS= 30 cycles
Methods
© 2011 The Bode Technology Group, Inc.
Results- Peak Height Ratio
vs RFU
• At which RFU value does the majority of the peak height ratios fall below our expected balance – ID+= 60%
– PP16 HS= 50%
– Using an analytical threshold of 75 RFU
• Plotted PHR v Avg RFU for 50, 100, 150, 200pg
• Zoom in on PHR below expected balance % – Heights could be considered unreliable under this
value and drop out may be possible
© 2011 The Bode Technology Group, Inc.
Results Comparison of Segnal Strength with Peak Height Ratio for Identifiler Plus 25ul Reactions at
28 cycles using 25, 50, 100 and 200 pg of Template DNA
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 100 200 300 400 500 600 700
Average Peak Height per Allele (RFU)
Pea
k H
eig
ht
Rat
io (
Max
/Min
)
Comparison of Signal Strength with Peak Height Ratio for Identifiler Plus
25uL Reactions at 28 cycles using 25, 50, 100 and 200 pg of Template DNA
© 2011 The Bode Technology Group, Inc.
Results Comparison of Signal Strength with Peak Height Ratio for PowerPlex 16 HS
25uL Reactions at 28 cycles using 25, 50, 100 and 200 pg of Template DNA Comparison of Signal Strength with Peak Height Ration for PowerPlex 16 HS 25ul Reactions
at 30 amplification cycles using 25, 50, 200 and 200 pg of Template DNA
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 50 100 150 200 250 300 350 400
Average Peak Height per Allele (RFU)
Pea
k H
eig
ht
Rat
io (
min
pea
k/m
ax p
eak)
© 2011 The Bode Technology Group, Inc.
Results Summary
• Table shows highest peak threshold observed
with sister allele < analytical threshold – Again, with ABI’s ID+, decreasing analytical threshold means
that you can decrease stochastic threshold
– Promega’s PP16 HS stochastic threshold not affected by
changing analytical threshold
Analytical Threshold 50RFU 75RFU
ID + 158 222
PP16 HS 129 129
© 2011 The Bode Technology Group, Inc.
Comparison of ID/ID+ and
PP16/PP16 HS
• ID+
– Greater stochastic threshold than ID (250RFU vs 200RFU)
– Other studies performed by Bode confirmed ID+ is more
sensitive, which could aid interpretation
• PP16 HS
– Decreased stochastic threshold than PP16 (200RFU vs
250RFU)
– Other studies performed by Bode confirmed PP16 HS is only
slightly more sensitive, which probably will not greatly aid
interpretation
© 2011 The Bode Technology Group, Inc.
Summary
• Data from 2009 study examining four kits proved to be solid basis for study this year on two additional kits – Stochastic thresholds still in same 200-250rfu range
• Work in progress – Looking into increasing PP16 stochastic threshold to 250 rfu also
– Use in mixture interpretation software for HTP lab (need macros/software!!!)
– Use of different types of statistics to allow use of more loci
© 2011 The Bode Technology Group, Inc.
Recommendations
• Single source profile with high level of
heterozygosity
• Get data points from ~3-5 individuals
• Plot PHR vs Avg RFU
© 2011 The Bode Technology Group, Inc.
Many Thanks!
• Jon Davoren, Bode
• Katherine Butler, GWU
• Hallie Garofalo, Bode (VCU)
• Mike Cariola, Bode
• Amy Jeanguenat, Bode
Stephanie.Sivak@bodetech.com
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