yfiler®plus verification test site results
DESCRIPTION
Author: Lynne K. Burley, Santa Clara County Crime Laboratory, San Jose, CA Overview: - Current limitations - Yfiler Plus design and configuration - VTS test site data Sensitivity -- Inhibition -- Mixtures -- Non-probative casework -- Discrimination of close relativesTRANSCRIPT
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Lynne K. Burley Santa Clara County Crime Laboratory
San Jose, CA
Yfiler® Plus: Verification Test Site Results
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Agenda
• Current limitations • Yfiler Plus design and configuration • VTS test site data
– Sensitivity – Inhibition – Mixtures – Non-probative casework – Discrimination of close relatives
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Challenges to overcome Male relatives cannot be excluded
Adventitious matches in the database
Limited ability to overcome inhibition
Limited ability to amplify degraded samples
Lengthy amplification time
Allelic ladder does not cover full spectrum of known alleles
Taq
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Yfiler Plus kit configuration
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Streamlined workflow Reaction
component Volume per reaction (uL)
Yfiler Plus
Volume per reaction (uL)
Yfiler
Master Mix
10 9.2
Primer 5 5
Sample 10 10
Taq Included in master mix 0.8
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Reduced amplification time Yfiler
95°C-11 min
94°C-1 min
61°C-1 min x 30 cycles
72°C-1 min
60°C-80 min
~3 hours
Yfiler Plus
95°C-1 min
94°C-4 sec
61.5°C-1 min
60°C-22 min
~1.5 hours
x 30 cycles
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Expanded allelic ladder
Kit Alleles Virtual Bins Yfiler 137 72
Yfiler Plus 345 179
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VTS plate setup
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VTS sensitivity experiment
• Two-fold serial dilution of Control DNA 007 (0.1ng/uL*): 1ng, 500pg, 250pg, 125pg, 62.5pg, 31.25pg
• 10uL of each dilution added to reaction • Replicates of four • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu * - 007 provided actually quant’d at 0.07ng/uL
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Average total peak height - sensitivity experiment
0
5000
10000
15000
20000
25000
30000
35000
31.25pg 62.5pg 125pg 250pg 500pg 1ng
Concentration
rfu Yfiler Plus
Yfiler
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Percent of alleles detected - sensitivity study
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 20 40 60 80 100 120 140
Concentration (pg)
% A
llele
s D
etec
ted
YfilerYfiler Plus
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Intracolor balance - sensitivity study
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
250pg Yfiler Plus 250pg Yfiler 500pg Yfiler Plus 500pg Yfiler 1ng Yfiler Plus 1ng Yfiler
Concentration/kit
% b
alan
ce
ICB 6-FAM aveICB VIC aveICB NED aveICB TAZ/PET aveICB SID ave
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Sensitivity study - baseline
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VTS inhibition experiment • 1ng (10uL of 0.1ng/uL) control DNA 007
– 10ng/uL Humic Acid (25ng/uL?) – 100ng/uL Humic Acid (250ng/uL?) – 20uM Hematin (50uM?) – 200uM Hematin (500uM?)
• Replicates of four (Yfiler Plus) and two (Yfiler) • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
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VTS inhibition study – hematin Yfiler
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VTS inhibition experiment – hematin Yfiler Plus
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VTS inhibition experiment – humic acid Yfiler
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VTS inhibition experiment – humic acid Yfiler Plus
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Average intracolor balance with inhibitors
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
hematin20-YFP
hematin20-YF
hematin200-YFP
hematin200-YF
humicacid 10-
YFP
humicacid 10-
YF
humicacid100-YFP
humicacid
100-YF
1ngYFP
1ng YF
Inhibitor and kit
% b
alan
ce
ICB 6-FAMICB VICICB NEDICB TAZ/PETICB SID
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Inhibition – average peak height
500
1500
2500
3500
4500
5500
Ave
rage
rfu
Yfiler PlusYfiler
Hematin 20
Hematin 200
Humic acid 10
Humic acid 100
1ng
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VTS Male:male mixtures • Four mixture ratios (1:1, 1:2, 1:4, 1:8) amplified
in triplicate (Yfiler Plus) and duplicate (Yfiler) • All samples had ~20ng/uL of female background
DNA (200ng per sample) • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
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VTS Male:male mixtures • Major:minor consistent across all loci at 1:2, 1:4,
and 1:8 for Yfiler Plus • Major:minor flip-flopped at two loci in the Yfiler
1:2 mixtures
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VTS Male:female mixtures
• Two male:female mixture ratios (1:1000 and 1:4000) in replicates of three (Yfiler Plus) and two (Yfiler)
• 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
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Intracolor balance male:female mixtures
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1ng_
YFP
1ng_
YF
MF 1:10
00_Y
FP1
MF 1:10
00_Y
FP2
MF 1:10
00_Y
FP3
MF 1:10
00_Y
F1
MF 1:10
00_Y
F2
MF 1:40
00_Y
FP1
MF 1:40
00_Y
FP2
MF 1:40
00_Y
FP3
MF 1:40
00_Y
F1
MF 1:40
00_Y
F2
% b
alan
ce
ICB 6-FAMICB VICICB NEDICB TAZ/PETICB SID
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VTS male:female mixtures 1:1000 Yfiler
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VTS male:female mixtures 1:1000 Yfiler Plus
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VTS male:female mixtures 1:4000 Yfiler
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VTS male:female mixtures 1:4000 Yfiler Plus
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VTS male:female mixtures • Known artifact observed in TAZ dye
channel
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VTS non-probative samples
• n=60 – NIST 2391b dilution series – Proficiency test samples – Adjudicated case samples with “anomolies” – Paternal references from six families (n=26),
half rxns • Amp target same as previous or 0.75ng • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
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Null allele and allele out of bin
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Null allele and allele out of bin
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Out of bin alleles
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Out of bin alleles…and a duplication
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Adjudicated sexual assault
• Case background • Quant of victim blood reference –
186ng/uL (human)/(6.4ng/uL) male • Autosomal results • Y-STR testing • Analysis of victim buccal swab
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Adjudicated sexual assault
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Adjudicated sexual assault
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Adjudicated sexual assault
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Family study
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Family study
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Family study
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Major improvements Male relatives cannot be excluded
10 new Y-STR loci including 7 randomly mutating Y-STRS
Adventitious matches in the database Limited ability to overcome inhibition
Improved inhibitor resistance
Limited ability to amplify degraded samples
11 mini STRs below 220bp
Lengthy amplification time
Shorter amplification time
Allelic ladder does not cover full spectrum of known alleles
Expanded allelic ladder
Taq Eliminated